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DK2978444T3 - CANCERVACCINE FOR DOGS - Google Patents

CANCERVACCINE FOR DOGS Download PDF

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DK2978444T3
DK2978444T3 DK14716530.2T DK14716530T DK2978444T3 DK 2978444 T3 DK2978444 T3 DK 2978444T3 DK 14716530 T DK14716530 T DK 14716530T DK 2978444 T3 DK2978444 T3 DK 2978444T3
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Demoyen Pierre Langlade
Simon Wain-Hobson
Christelle Liard
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Invectys
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Description

DESCRIPTION
[0001] The present invention relates to cancer vaccination in dogs.
Background of the invention: [0002] Like their human counterparts, dogs that live in developed countries have seen their life expectancy consistently prolonged. Therefore, the global burden of cancers continues to increase largely because of the aging and growing dog population.
[0003] The incidence rate of cancers in the canine population is estimated to be between 282.2 to 958 per 100,000 dogs (Merlo et al. 2008, Vascellari et al. 2009). The most frequent tumors in dogs are mammary tumors in females (70.5% of all cancers), non-Hodgkin's lymphomas (8.4% in females and 20.1% in males) and skin tumors (4% in females and 19.9% in males). Moreover, according to the European Society of Veterinary Oncology 50% of dogs over ten years are going to die from a cancer-related problem.
[0004] The panel of treatments available against veterinary cancer is substantially reduced compared with those available in human oncology.
[0005] Surgery remains the best way to treat animal tumors. This method presents the advantage of being accessible for many veterinarians, and, in many cases, it can be curative. However, to be curative, surgery must be bold. However in some cases the tumor is too large, too dispersed or just not accessible enough to be entirely removed. If not totally curative, surgery can still be a palliative solution to improve the animal's comfort and prolong its life expectancy.
[0006] Radiotherapy is another important means to treat certain types of cancers in the veterinary field. It is of particular interest for tumors which are hardly accessible for surgery like cerebral tumors (de Fornel et al. 2007). Furthermore, recent studies in humans have demonstrated that ionizing radiation (IR) could act as an immunomodulator by inducing substantial changes in the tumor microenvironment, including triggering an inflammatory process. Furthermore, the cost and the availability of the material make access to radiation therapy complicated for companion animals.
[0007] Chemotherapy is more and more used in animal oncology (Marconato 2011). Taking advantages of medical advances in human cancer therapy, there are more and more molecules available like vincristine, cyclophosphamide, carboplatin or cisplatin, to treat companion animals. In the veterinary field, anticancer drugs are particularly used in the treatment of tumors derived from hematopoietic tissue (lymphomas, leukemias). For example the CHOP protocol, combining cyclophosphamide, doxorubicin, vincristine and prednisone is currently used in the treatment of numerous lymphomas (Chun 2009). Chemotherapeutic agents can be particularly efficient in prolonging the life span of a cancerous animal from a few weeks to several months (the median survival time of dogs treated with the CHOP protocol is 13 months). Interestingly, the side effects dreaded by human patients, such as vomiting, diarrhea, hair loss, are usually less frequent in companion animals. Unfortunately, most of the time chemotherapy is not curative in pets and the tumor often escapes treatment.
[0008] Therefore, just as in human medicine, targeted therapies are in development in veterinary medicine. Thus, some drugs are already available in the clinics like "Masitinib", an inhibitor of the tyrosine kinase c-kit (Gentilini 2010). Other treatments, including immunotherapies, are under investigation (Manley et al. 2011). These immunotherapeutic treatments are all based on the fact that it is possible to activate the immune system of the host against cancer cells.
[0009] The relationship between the host immune system and cancer is dynamic and complex. Each type of tumor cell harbors a multitude of somatic mutations and epigenetically deregulated genes, the products of which are potentially recognizable as foreign antigens by immune cells (MUC-1, β-catenin, telomerase...) (Fridman et al. 2012). Growing tumors contain infiltrating lymphocytes called TILs (Tumor Infiltrating Lymphocytes). These killer cells are often ineffective at tumor elimination in vivo but can exert specific functions in vitro, that is to say outside the immunosuppressive tumor microenvironment (Restifo et al. 2012). This is because the tumor stroma contains many suppressive elements including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDCs); soluble factors such as interleukin 6 (IL-6), IL-10, vascular endothelial growth factor (VEGF), and transforming growth factor beta (TGFP) that down modulate antitumor immunity (Finn 2008, Hanahan and Weinberg 2011). Consequently, the choice of a pertinent tumor associated antigen (TAA) and the bypass of cancer associated immunosuppression are two critical points for a therapeutic vaccine to succeed (Disis et al. 2009).
[0010] Recent introduction of active cancer immunotherapy (also referred to cancer vaccines) in the clinical cancer practice emphasizes the role of immune responses in cancer prognosis and has led to a growing interest to extend this approach to several human and companion animal cancers (Dillman 2011, Topalian et al. 2011) (Jourdier et al. 2003).
[0011] Peruzzi et al, 2010, Vaccine, 28(5):1201-1208 discloses an immunogenic composition comprising a nucleic acid that comprises a dog telomerase reverse transcriptase (dTERT) deprived of telomerase catalytic activity. Peruzzi et al, 2010, Molecular Therapy, 18(8):1559-1567, discloses the use of this composition for treating dogs affected with B-cell lymphoma.
[0012] In this context, there is still a need for an innovative cancer vaccine strategy for dogs, which would overcome the challenge of breaking tolerance and inducing an immune response in the animal.
Summary of the invention: [0013] The inventors now propose a cancer vaccine strategy for dogs, based on the telomerase reverse transcriptase (TERT).
[0014] A subject of the invention is thus an immunogenic composition comprising a nucleic acid that comprises a sequence encoding a dog telomerase reverse transcriptase (TERT) wherein the dog TERT is deleted of amino acids VDD which provides inactivation of telomerase catalytic activity, and is further deleted of N-terminal 47 amino acids with respect to full-length dog TERT sequence.
[0015] The nucleic acid is preferably DNA, preferably in form of a plasmid.
[0016] In a particular embodiment, the nucleic acid further comprises a non-dog TERT antigenic fragment.
[0017] A further subject of the invention is a nucleic acid that comprises a sequence encoding a dog telomerase reverse transcriptase (TERT) wherein the dog TERT is deleted of amino acids VDD which provides inactivation of telomerase catalytic activity, and is further deleted of N-terminal 47 amino acids with respect to full-length dog TERT sequence, and optionally further comprises a non-dog TERT antigenic fragment.
[0018] The immunogenic composition or the nucleic acid is useful in triggering an immune response in a dog, against cells that over-express telomerase, such as dysplasia cells or tumor cells.
[0019] The immunogenic composition or the nucleic acid is thus particularly useful in treating a tumor in a dog, preferably by intradermal or intramuscular route.
[0020] Such treatment can be referred to as an active immunotherapy or a therapeutic vaccination, as it triggers an immune response against the tumor, especially a cytotoxic CD8 T cell response, along with a specific CD4 T cell response.
[0021] The invention makes it possible to induce dTERT specific responses in dogs with neoplasias and so can be used for immunotherapeutic treatments of the neoplasias in a clinical setting.
The invention is also useful to induce dTERT specific responses in healthy dogs that could be at risk for cancer, e.g. by genetic predisposition, or in healthy dogs from a certain age (e.g. more than 10 years, preferably more than 12 years old), so as to prevent the onset of cancer.
[0022] Generally speaking, the treatment of the invention may induce long term immune memory responses in healthy dogs, dogs at risk of developing a cancer and those presenting a cancer.
Brief description of the Figures: [0023]
Figure 1A shows pDUV5 nucleotide sequence (SEQ ID NO: 1) and corresponding amino acid sequence comprising dog TERT (dTERT) amino acid sequence (SEQ ID NO: 2).
The plasmid pDUV5 encodes a near full length dog TERT nucleotide sequence. The nucleotide sequence encoding 3 key amino acids in the catalytic site of the protein have been deleted (VDD). Moreover, the sequence controlling the importation into the nucleoli (Nucleolar addressing signal) has been deleted (nucleotide sequence encoding 47 first Amino Acids in the N-term sequence of dTERT protein). Moreover the DNA sequence encoding the human ubiquitin has been added upstream the dTERT sequence. Presence of the ubiquitin protein enhances the addressing of the dTERT protein to the proteasome and increases class I presentation of derived peptides. However, as the human and dog ubiquitin sequences are identical at the protein level, there is no biological incompatibility. Downstream the dTERT sequence, the sequence of the V5 peptide of the flu was inserted to facilitate the detection of the protein.
Nucleotides 1-6 Hindlll restriction site for subcloning
Nucleotides 13-240 ubiquitin
Nucleotides 241-3459 dog TERT
Nucleotides 2670-2671 inactivating deletion of 9 bp encoding VDD residues
Nucleotides 3460-3513 influenza A A2 epitope
Nucleotides 3514-3555 SV5 V5 tag
Nucleotides 3556-3561 two stop codons
Nucleotides 3562-3567 Xbal restriction site for subcloning
Figure 1B shows pCDT nucleotide sequence (SEQ ID NO: 3) and corresponding amino acid sequence containing cat/dog hybrid TERT amino acid sequence (SEQ ID NO:4).
The plasmid pCDT encodes the cat/dog hybrid TERT (hyTERT) comprising 54.4% from the cat TERT and 35.9% from the dog TERT sequence. The nucleotide sequence encoding 3 key amino acids in the catalytic site of the protein have been deleted (VDD). Moreover, the sequence controlling the importation into the nucleoli (Nucleolar addressing signal) has been depleted (nucleotide sequence encoding 47 first Amino Acids in the Nter sequence of hyTERT protein). The DNA sequence encoding the human ubiquitin has been added upstream the hyTERT sequence. The presence of the ubiquitin protein enhances the addressing of the hyTERT protein to the proteasome and increases class I presentation of the derived peptides. Downstream the hyTERT sequence, the sequence of the V5 peptide of the flu was inserted to facilitate the detection of the protein.
Nucleotides 1-6 Hindlll restriction site for subcloning
Nucleotides 13-240 ubiquitin
Nucleotides 241-1413 dog TERT (35.9 % of TERT sequences)
Nucleotides 1414-3351 cat TERT (54.4% of TERT sequences)
Nucleotides 3352-3456 dog TERT last exon
Nucleotides 3457-3510 influenza A2 epitope
Nucleotides 3511-3552 SV5 V5 tag
Nucleotides 2667-2668 inactivating deletion of 9 bp encoding VDD residues
Nucleotides 3553-3558 two stop codons
Nucleotides 3559-3564 Xbal restriction site for subcloning
Figure 2A shows a simplified map of pcDNA3.1 expression plasmid into which the dog or hybrid TERT nucleic acid sequences are cloned.
Figure 2B shows dog TERT protein sequence (SEQ ID NO: 5). The region covered by the dTERT 15mer peptide pool overlapping by 11 residues (70 peptides in total) that is used for in vitro immunization studies and ELIspot assays in dog PBMCs is shown in grey.
Figure 3 shows that pDNA constructs are safe (Trapeze). Lysates obtained from CrFK cells transfected with hTERT, pCDT, or pDUV5 plasmids were analyzed for telomerase activity by the TRAP assay. The level of telomerase activity is shown as relative telomerase activity compared with that of control template measured in each kit and with the activity of a wild type human telomerase (hTERT). All samples at 2.1 pg protein concentration were measured in triplicate, error bars are standard error of the mean (SEM), (**P=0.0032, hTERT vs pDUV5 unpaired t test).
Figure 4 is a graph showing that mice immunized with pDUV5 mount specific interferon-y-secreting CD8 T-cell responses against H2 restricted dog TERT peptides. 7 week-old C57/B16 female mice were immunized intradermally (ID) or intramuscularly (IM) (10 mice per group) with 100 pg pDUV5 plasmid at day 0 and boost 14 days later. At the same time, control mice received PBS via ID or IM route (6 mice per group). Ten days after boost, spleens of all mice were harvested. Splenocytes were Ficoll purified and stimulated in triplicates with 5 pg/mL of relevant class I peptides (p580, p621 or p987) for 19 hours. Spots were revealed with a biotin-conjugated detection antibody followed by streptavidin-AP and BCIP/NBT substrate solution. Results are the mean ± standard deviation. Mann Whitney non parametric test, * p-value < 0.05, **: p-value < 0.01.
Figures 5A and 5B show that ID or IM immunization of mice with pDUV5 plasmid results in a dog TERT specific cytotoxic T-lymphocyte (CTL) response measurable in vivo by elimination of transferred target cells which were pulsed with dog TERT peptides restricted to H2.
Seven week-old C57/B16 female mice were immunized intradermally (ID) or intramuscularly (IM) with 100 pg pDUV5 plasmid at day 0 and day 14 post-priming. At day 9 post-boost injection, syngeneic splenocytes, pulsed with individual dTERT peptides restricted to H2 (either p987 or p621) or left unpulsed were labeled with carboxyfuorescein-diacetate succinimidyl ester (CFSE) at three different concentrations: high = 1 pM (p621), medium = 0.5 pM (p987) and low = 0.1 pM (unpulsed). The same number of high, medium or low CFSE labeled cells was transferred IV to vaccinated mice. After 15-18 hours, the disappearance of peptide-pulsed cells was determined by fluorescence-activated cell-sorting analysis in the spleen. The percentage of specific lysis was calculated by comparing the ratio of pulsed to un-pulsed cells in vaccinated versus control mice. 1. (A) Example of the in vivo CTL assay showing the elimination of target cells pulsed with p987 (medium, M)/ or p621 peptide (High, H) in the spleen of mice injected via the ID route (left panel). No such disappearing is observed in control mice injected ID with PBS IX (right panel). H= high, M= Medium, L= Low 2. (B) Percentage of specific lysis for each mouse against each individual peptide in the spleen after IM or ID vaccination with pDUV5. Horizontal bars show average percentage of lysis per peptide and per immunization route. Standard deviations are also plotted. Representative data from 2 independent experiments (n = 10 individual animals/group). Kruskal-Wallis analysis with Dunn's multiple comparison test, ns: not significant. Statistical significance is set at p-value < 0.05.
Figures 6A and 6B show IFNy+specific CD8 and CD4 T-cell responses against H2 restricted hyTERT peptides in mice immunized with pCDT.
Seven week-old female mice were immunized intradermally (ID) or intramuscularly (IM) with either 100 pg pCDT plasmid or PBS at day 0 and boost 14 days later. Ten day post-boost, spleens were harvested. Splenocytes were Ficoll-purified and stimulated in triplicates with 5 pg/mL of relevant peptides for 19 hours. Spots were revealed with a biotin-conjugated detection antibody followed by streptavidin-AP and BCIP/NBT substrate solution. 1. (A) Plasmid vaccinated groups were composed of five C57/B16 mice, and control groups, of three mice. Splenocytes were stimulated with class I peptides p580, p621 and p987. Results show the frequency of peptide specific IFN-γ producing CD8 T cells. 2. (B) Plasmid vaccinated groups were composed of 9 Balb/cBy mice immunized IM and 5 ID. Control groups of 8 Balb/cBy mice injected IM and 4 ID. Splenocytes were stimulated with class II peptides p951, p1105, p1106 and p1109. Results show the frequency of peptide specific IFN-γ producing CD4 T cells.
Results are the mean ± standard deviation. Mann Whitney non parametric test, * p-value < 0.05, **: p-value < 0.01.
Figures 7A and 7B show hyTERT specifc cytotoxic T-lymphocyte (CTL) response in mice immunized with pCDT plasmid, measurable in vivo by elimination of transferred target cells which were pulsed with hybrid TERT peptides restricted to H2. 7 week-old C57/B16 female mice were immunized ID or IM with 100 pg pCDT plasmid at day 0 and day 14 post-priming. At day 9 post-boost injection, syngeneic splenocytes, pulsed with individual dTERT peptides restricted to H2 (either p987 or p621) or left unpulsed were labeled with carboxyfluorescein-diacetate succinimidyl ester (CFSE) at three different concentrations: high = 1 μΜ (p987), medium = 0.5 pM (p621) and low = 0.1 pM (unpulsed). The same number of high, medium or low CFSE labeled cells was transferred IV to vaccinated mice. After 15-18 hours, the disappearance of peptide-pulsed cells was determined by fluorescence-activated cell-sorting analysis in the spleen. The percentage of specific lysis was calculated by comparing the ratio of pulsed to un-pulsed cells in vaccinated versus control mice. 1. (A) Example of the in vivo CTL assay showing the elimination of target cells pulsed with p621 peptide (High, H) or p987 peptide (Medium, M) in the spleen of a mouse vaccinated ID (left panel) with pCDT. No such disappearing is observed in control mice injected ID with PBS IX (right panel). 2. (B) Percentage of specific lysis for each mouse against each individual peptide in the spleen after IM or ID vaccination with pCDT. Horizontal bars show average percentage of lysis per peptide and per immunization route. Standard deviations are also plotted. Representative data from 2 independent experiments (n = 10 individual animals/group). Kruskal-Wallis analysis with Dunn's multiple comparison test, * p< 0,1, *** p<0,001, ns: not significant. Statistical significance is set at p-value < 0.05.
Figure 8 shows principle of in vitro immunization in dog PBMCs
Frozen dog PBMCs were incubated with recombinant canine GM-CSF (rcGM-CSF) and canine IL-4 (rclL-4) or human FIT3 (hFIT3) ligand for 24h. Maturation stimuli (rcTNFa, hIL-7 and rcIL-1 β) were then added with dTERT overlapping peptides pools for 3 days. Eleven or 18 days of culture were performed and, TERT specific T cells were then detected via an IFN-γ ELISpot assay.
Figures 9A and 9B show a repertoire of dTERT specific IFN- γ secreting T cells in PBMCs from a naive dog
Frozen PBMCs incubated during 24 hours with either rcGM-CSF and rclL-4 or hFIT3 ligand and matured 3 day long with dTERT overlapping peptides pools and maturation cytokines (rcTNFa, hlL-7 and calL-1 β) were harvested after 11 or 18 days of culture to perform an ELISpot IFN-γ. Results show the frequency of peptide specific IFN-γ producing T cells/106 canine PBMC after 11 days (A) or 18 days (B) of culture.
Figures 10A and 10B show the kinetics of the specific ΙΡΝγ T cell response against a first pool of dTERT peptides (pool 6), and a second pool of dTERT peptides (pool 19), respectively.
Six naive beagle healthy dogs were injected intradermally with 400 pg of pDUV5 DNA followed by electroporation, at days 0, 29, 57 and 142. Peripheral blood was drawn and mononuclear cells tested for dog telomerase specific peptides belonging either to pool 6 or pool 19 pepitdes according to the method of Martinuzzi et al., 2011. ΙΡΝγ specific T cell responses were detected by ELISPOT assay, for pool 6 and 10 dTERT peptides, all of which above baseline readings.
Figures 11A and 11B show the kinetics of the specific ΙΡΝγ T cell response against pool 6 dTERT peptides, and pool 10 dTERT peptides, respectively. pDUV5 DNA vaccination at days 57 and 142 show classical long term memory responses, that is rising sharply and decaying more slowly.
Figure 12 shows that tumor bearing dogs and healthy dogs have dTERT specific T lymphocytes (pool 4 peptides). Peripheral blood was drawn and in vitro stimulation protocol was performed as described Martinuzzi zt al, 2011.
Detailed description of the invention:
Definitions [0024] The telomerase consists of an RNA template and protein components including a reverse transcriptase, designated "Telomerase Reverse Transcriptase" (TERT), which is the major determinant of telomerase activity. Unless otherwise specified, in the present specification, the term "telomerase" refers to TERT.
[0025] In the present invention, the term "dog TERT" refers to the TERT sequence of any domestic dog (also designated Canis familiaris or Canis lupus familiaris). A dog TERT mRNA sequence is available with NCBI accession number NM_001031630 (XM_545191). Dog TERT amino acid sequence is shown as SEQ ID NO:5.
[0026] The invention can make also use of non-dog telomerase (TERT) sequence, which can be from any human or non-human mammal, e.g. from cat. The term "cat TERT" refers to the TERT sequence of any domestic cat (also designated as Felis catus or Felis silvestris catus). Partial molecular cloning of the cat TERT gene (237 bp of mRNA) has been reported by Yazawa et al. 2003. The inventors herein provide a longer sequence of Felis catus TERT. The corresponding amino acid sequence is shown as SEQ ID NO:7.
[0027] The "telomerase catalytic activity" refers to the activity of TERT as a telomerase reverse transcriptase. The term "deprived of telomerase catalytic activity" means that the nucleic acid sequence encodes a mutant TERT, which is inactive.
[0028] The term "hybrid" or "chimeric" amino acid or nucleotide sequence means that part of the sequence originates from one animal species and at least another part of the sequence is xenogeneic, i.e. it originates from at least one other animal species.
[0029] When referring to a protein, the term "fragment" preferably refers to fragment of at least 10 amino acids, preferably at least 20 amino acids, still preferably at least 30, 40, 50, 60, 70, 80 amino acid fragments.
[0030] In the context of the invention, the term "antigenic fragment" refers to an amino acid sequence comprising one or several epitopes that induce T cell response in the animal, preferably cytotoxic T lymphocytes (CTLs). An epitope is a specific site which binds to a T- cell receptor or specific antibody, and typically comprises about 3 amino acid residues to about 30 amino acid residues, preferably 8 or 9 amino acids as far as class I MHC epitopes are concerned, and preferably 11 to 25 amino acids as far as class II MHC epitopes are concerned.
[0031] The term "immunogenic" means that the composition or construct to which it refers is capable of inducing an immune response upon administration (preferably in a dog). "Immune response" in a subject refers to the development of a humoral immune response, a cellular immune response, or a humoral and a cellular immune response to an antigen. A "humoral immune response" refers to one that is mediated by antibodies. A "cellular immune response" is one mediated by T-lymphocytes. It includes the production of cytokines, chemokines and similar molecules produced by activated T-cells. Immune responses can be determined using standard immunoassays and neutralization assays for monitoring specifically the humoral immune response, which are known in the art. In the context of the invention, the immune response preferably encompasses stimulation or proliferation of cytotoxic CD8 T cells and/or CD4 T cells.
[0032] As used herein, the term "treatment" or "therapy" includes curative treatment. More particularly, curative treatment refers to any of the alleviation, amelioration and/or elimination, reduction and/or stabilization (e.g., failure to progress to more advanced stages) of a symptom, as well as delay in progression of the tumor or dysplasia or of a symptom thereof.
[0033] As used herein, the term "prevention" or "preventing" refers to the alleviation, amelioration and/or elimination, reduction and/or stabilization (e.g., failure to progress to more advanced stages) of a prodrome, i.e. any alteration or early symptom (or set of symptoms) that might indicate the start of a disease before specific symptoms occur.A cell that "overexpresses telomerase" refers to a cell in a subject, which either expresses telomerase, e.g. upon mutation or infection, whereas it does usually not, under normal conditions, or to a cell in a subject which expresses a higher level of telomerase (e.g. upon mutation or infection), when compared to normal conditions. Preferably the cell that overexpresses telomerase shows an increase of expression of at least 5%, at least 10%, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more.
Nucleic acid constructs [0034] It is herein described a nucleic acid that comprises a sequence encoding (i) a dog telomerase reverse transcriptase (TERT) deprived of telomerase catalytic activity, or (ii) a fragment thereof.
[0035] The nucleic acid may be DNA or RNA, but is preferably DNA, still preferably double stranded DNA.
[0036] As a first safety key, the TERT sequence is deprived of telomerase catalytic activity. The sequence that encodes dog TERT contains mutations that provide inactivation of the catalytic activity, namely a deletion of amino acids VDD, as shown on Figure 1A or 1B.
As a second safety key, the sequence encoding dog TERT is further deprived of a nucleolar localization signal. This nucleolar localization signal is correlated with the enzymatic activity of TERT. This signal corresponds to the N-terminal 47 amino acids at the N-terminus of the TERT sequence.
Preferably the sequence encoding dog TERT is deleted of N-terminal 47 amino acids with respect to the full-length dog TERT sequence.
Dog TERT sequence deleted of amino acids VDD and of the N-terminal 47 amino acids is shown as SEQ ID NO: 6.
[0037] It is herein disclosed that the nucleic acid may encode dog TERT sequence or a fragment thereof only, which preferably corresponds to at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% of the dog TERT sequence deleted of the N-terminal 47 amino acids.
Preferably the nucleic acid encodes dog TERT amino acid sequence comprising, or consisting of, SEQ ID NO: 5 or SEQ ID NO: 6.
[0038] The nucleic acid may further encode a non-dog TERT antigenic fragment. This embodiment is preferred, to favor breakage of tolerance towards a self-antigen, and induce an efficient immune response along, with an immune memory response in the dog. The presence of non-dog TERT fragment(s) advantageously engages certain subtypes of CD4+ T cells, providing help for antitumor immunity, and reversing potential regulation by secreting certain cytokines called Th1 cytokines.
[0039] The dog and non-dog TERT sequences or fragments thereof are preferably fused, to be expressed as a hybrid or chimeric protein. Alternatively, the dog and non-dog TERT sequences or fragments thereof may be separated, but carried on the same vector, e.g. the same plasmid.
[0040] Preferably the non-dog TERT antigenic fragment corresponds to a fragment absent or eliminated from the dog TERT sequence, to the extent it does not complement the loss of catalytic activity or the loss of the nucleolar localization signal.
[0041] The dog TERT sequence, or fragment thereof, can represent at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% of all TERT sequences in the nucleic acid, plasmid, or other vector.
The dog TERT sequence or fragment may represent at least 90% of the hybrid or chimeric TERT protein.
The dog TERT sequence or fragment may represent at least 60% of the hybrid or chimeric TERT protein.
The non-dog TERT antigenic fragment preferably originates from a cat TERT sequence.
The non-dog TERT antigenic fragment is advantageously processed by dendritic cells, thereby generating CD4 T cell help.
[0042] It is described a nucleic acid that encodes a protein sequence selected from the group consisting of SEQ ID NO: 2, 4, 5 or 6.
Such nucleic acid may comprise a sequence selected from the group consisting of SEQ ID NO:1 or 3, or nucleotides 241-3459 of SEQ ID NO: 1, or nucleotides 241-1413 or 241-1407or nucleotides 3352-3456 or 3298-3456 of SEQ ID NO: 3.
[0043] In a particular embodiment, the nucleic acid may further encode a protein which enhances the addressing of the TERT protein to the proteasome and increases class I presentation of the derived peptides. Said protein may be preferably ubiquitin, or it may be any chaperon protein, e.g. calreticulin.
Genetic constructs, immunogenic compositions and administration [0044] Preferably, the nucleic acid is a genetic contrast comprising a polynucleotide sequence as defined herein, and regulatory sequences (such as a suitable promoter(s), enhancer(s), terminator(s), etc.) allowing the expression (e.g. transcription and translation) of the protein product in the host cell or host organism.
[0045] The genetic constructs of the invention may be DNA or RNA, and are preferably doublestranded DNA. The genetic constructs of the invention may also be in a form suitable for transformation of the intended host cell or host organism, in a form suitable for integration into the genomic DNA of the intended host cell or in a form suitable for independent replication, maintenance and/or inheritance in the intended host organism. For instance, the genetic constructs of the invention may be in the form of a vector, such as for example a plasmid, cosmid, YAC, a viral vector or transposon. In particular, the vector may be an expression vector, i.e. a vector that can provide for expression in vitro and/or in vivo (e.g. in a suitable host cell, host organism and/or expression system).
[0046] In a preferred but non-limiting aspect, a genetic construct of the invention comprises i) at least one nucleic acid of the invention; operably connected to ii) one or more regulatory elements, such as a promoter and optionally a suitable terminator; and optionally also iii) one or more further elements of genetic constructs such as 3'- or 5'-UTR sequences, leader sequences, selection markers, expression markers/reporter genes, and/or elements that may facilitate or increase (the efficiency of) transformation or integration.
[0047] In a particular embodiment, the genetic construct can be prepared by digesting the nucleic acid polymer with a restriction endonuclease and cloning into a plasmid containing a promoter such as the SV40 promoter, the cytomegalovirus (CMV) promoter or the Rous sarcoma virus (RSV) promoter. In a preferred embodiment, the TERT nucleic acid sequences are inserted into a pcDNA3.1 expression plasmid (see Figure 2A).
Other vectors include retroviral vectors, lentivirus vectors, adenovirus vectors, vaccinia virus vectors, pox virus vectors, adenovirus-associated vectors and measle virus vectors.
[0048] Compositions can be prepared, comprising said nucleic acid or vector. The compositions are immunogenic. They can comprise a carrier or excipients that are suitable for administration in dogs (i.e. non-toxic, and, if necessary, sterile). Such excipients include liquid, semisolid, or solid diluents that serve as pharmaceutical vehicles, isotonic agents, stabilizers, or any adjuvant. Diluents can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin, among others. Any adjuvant known in the art may be used in the vaccine composition, including oil-based adjuvants such as Freund's Complete Adjuvant and Freund's Incomplete Adjuvant, mycolate-based adjuvants, bacterial lipopolysaccharide (LPS), peptidoglycans, proteoglycans, aluminum hydroxide, saponin, DEAE-dextran, neutral oils (such as miglyol), vegetable oils (such as arachis oil), Pluronic® polyols.
[0049] The nucleic acid or composition can be administered directly or they can be packaged in liposomes or coated onto colloidal gold particles prior to administration. Techniques for packaging DNA vaccines into liposomes are known in the art, for example from Murray, 1991.
[0050] Similarly, techniques for coating naked DNA onto gold particles are taught in Yang, 1992, and techniques for expression of proteins using viral vectors are found in Adolph, 1996.
[0051] For genetic immunization, the vaccine compositions are preferably administered intradermally, subcutaneously or intramuscularly by injection or by gas driven particle bombardment, and are delivered in an amount effective to stimulate an immune response in the host organism. In a preferred embodiment of the present invention, administration comprises an electroporation step, also designated herein by the term "electrotransfer", in addition to the injection step (as described in Mir 2008, Sardesai and Weiner 2011).
[0052] The compositions may also be administered ex vivo to blood or bone marrow-derived cells using liposomal transfection, particle bombardment or viral transduction (including cocultivation techniques). The treated cells are then reintroduced back into the subject to be immunized.
[0053] While it will be understood that the amount of material needed will depend on the immunogenicity of each individual construct and cannot be predicted a priori, the process of determining the appropriate dosage for any given construct is straightforward. Specifically, a series of dosages of increasing size, starting at about 5 to 30 pg, or preferably 20-25 pg, up to about 500 pg for instance, is administered to the corresponding species and the resulting immune response is observed, for example by detecting the cellular immune response by an ΙΡΝγ Elispot assay (as described in the experimental section), by detecting CTL response using a chromium release assay or detecting CD4 T cell (helper T cell) response using a cytokine release assay.
[0054] In a preferred embodiment, the vaccination regimen comprises one to three injections, preferably repeated three or four weeks later.
In a particular embodiment, the vaccination schedule can be composed of one or two injections followed three or four weeks later by at least one cycle of three to five injections.
In another embodiment, a primer dose is composed of one to three injections, followed by at least a booster dose every year, or every two or years for instance.
Prevention and treatment of tumors [0055] The nucleic acid or immunogenic composition as described above is useful in a method for preventing or treating a tumor in a dog. A method for preventing or treating a tumor in a dog is described, which method comprises administering an effective amount of said nucleic acid or immunogenic composition in a dog in need thereof. Said nucleic acid or immunogenic composition is administered in an amount sufficient to induce an immune response in the dog.
The tumor may be any undesired proliferation of cells, in particular a benign tumor or a malignant tumor, especially a cancer.
The cancer may be at any stage of development, including the metastatic stage. However preferably the cancer has not progressed to metastases.
In particular the tumor may be selected from the group consisting of bladder cancer, brain tumor, liver tumor, mammary tumors and carcinoma, mast cell tumors, malignant histiocytosis and histocytic sarcomas, squamous cell carcinomas, hemangiosarcoma, lymphoma, in particular B-cell lymphoma, melanoma, bone tumors (osteosarcoma), testicular tumors.
[0056] In a particular embodiment, the vaccination according to the invention may be combined with conventional therapy, including chemotherapy, radiotherapy or surgery. Combinations with adjuvant immunomodulating molecules such GM-CSF or IL-2 could also be useful.
[0057] The Figures and Examples illustrate the invention without limiting its scope. EXAMPLES: [0058] The inventors have constructed DNA vaccines encoding an inactivated form of dog TERT and a cat/dog hybrid TERT (Example 1), and have assessed their functionality, safety and immunogenicity.
They have demonstrated that the plasmids were correctly processed in vitro after transfection in mammalian cells and that the plasmid product of expression (TERT protein) was well expressed. Moreover, no enzymatic activity was detected and TERT proteins were found excluded for the transfected cells nucleoli, which evidences safety of the constructs (Example 2).
Then, the plasmids were found to be immunogenic and to elicit specific efficient CD8 T cells and CD4 T cells in mice (Example 3).
Example 1: Construction of the DNA plasmids [0059] In all constructs, the TERT sequence is preceded by a DNA sequence encoding the human-ubiquitin. The presence of the ubiquitin will increase the addressing of the TERT protein to the proteasome and increase the class I presentation pathway of TERT derived peptides. TERT sequence is followed by the sequence of the influenza protein V5 to facilitate future purification or detection of the fusion protein by Western Blot or histochemistry for example. The DNA sequence coding for the TERT protein has been deleted of 47 amino-acids in the N-Ter region, which encodes the nucleolar importation signal. Moreover, three aminoacids have been removed in the catalytic site of TERT (VDD), to inhibit the protein enzymatic activity. pDUV5 encodes the full-length of dog TERT nucleotide sequence, depleted of the N-term 47 amino acids (Figure 1A), pCDT encodes 54.4% of the cat TERT sequence and 35.9% of the dog TERT sequence (Figure 1B).
All TERT DNA sequences were synthetized from Genecust (Dudelange, Luxembourg). Then they were cloned into the pcDNA3,1 expression plasmid provided by Life technologies SAS (Saint-Aubin, France) using the Hindlll and Xbal restriction sites (see Figure 2A).. Plasmids were stored at -20°C, in PBS IX, at a concentration of 2 mg/mL prior use. The backbone plasmid was used as empty vector for western blot and Trap-Assay experiments. It consists of the pcDNA3.1 backbone plasmid deprived of the transgene protein DNA sequence (TERT).
Example 2: Functionality and safety of the plasmids: 2.1. Materials and methods
Cell culture [0060] The human 293T cell line used for transfection assays and immune-fluorescence experiments were kindly provided by Pr Simon Wain-Hobson (Pasteur Institute). The CrFK (Crandall-Reese feline kidney) cells used for the TRAP-assay were kindly provided by Pr J. Richardson (Ecole Vétérinaire de Maison Alfort). Cells were grown at 37°C, 5% CO2- in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated Fetal Calf Serum (FCS), 1% sodium-pyruvate, 1% penicillin-streptomycin pyruvate and 0.1% β-mercaptoethanol. All components of the culture medium were purchased from Life technologies SAS (Saint-Aubin, France).
Transfection assays [0061] Transfection of 293T cells were performed with either pCDT or pDUV5 plasmids using the JetPRIME® transfection kit (Polyplus-transfection SA, lllkirch, France) according to manufacturer's instruction. In a 6-well plate, 400 000 HeLa cells or 293T cells per well were seeded in 2 mL of DMEM culture medium, and cultured 24 hours at 37°C, 5% CO2 prior transfection. For each well, 2 pg of each plasmid diluted in 200 pL of jetPRIME® buffer, or 200 pL of jetPRIME® buffer only with respectively 4 pL of jetPRIME® agent were drop onto the cells. Transfection medium were removed 4 hours later and replaced by 2 mL of DMEM culture medium. Cells were put at 37°C, 5% CO2 and recovered for analysis 24 hours later.
Western Blots [0062] Transfected 293 T cells were lysed on ice with radioimmunoprecipitation assay (RIPA) lysis buffer (RIPA Buffer, Sigma Aldrich chimie SARL, Saint-Guentin Fallavier, France) containing protease inhibitors cocktail (Complete EDTA-free, Roche Diagnostic, Indianapolis, USA) for 10-20 minutes. Then, suspension was centrifuged 15 minutes at 14000 rpm at 4°C in order to remove cellular debris. The supernatants were harvested and the protein concentration was measured using the Bradford method. Protein samples were denatured 5 minutes at 95°C, separated on Nu-PAGE® Novex 4-12% Bis-Tris gels (Invitrogen, Carlsbad, USA) and transferred to PVDF membranes (iBIot® transfer stack, Invitrogen, Carlsbad,USA) using the iBIot® device (Invitrogen, Carlsbad, USA). The membrane was cut approximately at 60 kDa. First, the upper part membrane was probed with an anti-V5 antibody (Invitrogen, Carlsbad, USA) while the other part was probed with an anti-P-actin antibody (Sigma Aldrich chimie SARL, Saint-Quentin Fallavier, France), then samples were revealed by an ECL (Enhanced chemiluminescence) anti-mouse Horse Radish Peroxidase (HRP) linked antibody (GE Healthcare, Vélizy, France)). Immunoblot signals were reveled using 18 x 24 films and the corresponding cassette both products purchased from GE healthcare (Buckinghamshire, UK).
Immunofluorescence and microscopy [0063] Human 293T cells were seeded on 8-well Lab-Tek® chamber slides (Sigma Aldrich chimie SARL, Saint-Quentin Fallavier, France) at 20.103 cells/well in 200 pL of culture medium and incubated overnight at 37°C. The next day, culture medium was discarded. Ten pL of a mix solution containing 1 pg of either pCDT or PUF2 plasmid, 50 pL of OptiMEM (Life technologies SAS, Saint-Aubin, France) and 2.5 pL of Fugene HD (Promega France, Charbonnieres-les-bains, France) were added to the corresponding chamber. As control, 20.103 HeLa cells were incubated with the 10 pL of the same mix without plasmid. Chamber slides were left in the incubator for 24 hours. Transfected 293T cells were carefully washed with PBS IX and 200 μΙ_ 2% PFAwere added to each well for 10 minutes at +4°C, in order to fix and permeabilize the cells. Then wells were washed two times with PBS IX 0.05% Tween®20 and 293T cells were incubated 30 minutes at room temperature with 200 pL of Blocking solution (0.5% TritonX100; 3% BSA; 10% Goat Serum). Eventually, wells were incubated for 1.5 hours at room temperature with a primary mouse anti-V5 antibody (Life technologies SAS, Saint-Aubin, France) diluted in blocking solution at 1/200, with slight agitation. After three washes in PBS IX 0.05% Tween®20, a secondary goat anti-mouse-Alexa Fluor 488® antibody (Life technologies SAS, Saint-Aubin, France) diluted in blocking solution (1/500) was put in the wells for 45 minutes at room temperature away from light and under slight agitation. Wells were washed three times with PBS IX 0.05% Tween®20 and mounted with the Vectashield® mounting medium containing DAPI (Vector laboratories, Peterborough, UK). Slides were analyzed with a fluorescence microscope (Axio observer Z1, Carl Zeis Microimaging GmbH, Jena, Germany) equipped with an image processing and analysis system (Axiovision, Carl Zeis Microimaging GmbH, Jena, Germany). TRAP assay [0064] Telomerase activity was measured by the photometric enzyme immunoassay for quantitative determination of telomerase activity, utilizing telomeric repeat amplification protocol (TRAP) (Yang et al, 2002). CrFK (Crandell Rees Feline Kidney) telomerase-negative cells (Yazawa et al., 2003) were transfected with plasmids encoding pDUV5, or pCDT TERT constructs. As a positive control CrFK cells were transfected with a plasmid encoding the wild type human TERT (fully active). Briefly, 24 hours after transfection, CrFK cells were harvested by mechanical scraping and then washed twice with 1 mL PBS and pelleted by centrifugation 5 minutes at 3000g, at 4°C. Telomerase activity was assessed by TRAP-ELISA assay using the TeloTAGGG Telomerase PCR ELISAPLUS kit (Roche Diagnostics, Germany) according to the manufacturer's instructions. The protein concentration in the cell extract was measured by the Bradford method (Bio-Rad Laboratories). Three microliters of the cell extract (equivalent to 2.1, 0.21, 0.021 pg) was incubated in a Polymerase Chain reaction (PCR) mixture provided in the kit. The cycling program was performed with 30 minutes primer elongation at 25°C and then the mixture was subjected to 30 cycles of PCR consisting of denaturation at 94°C for 30 sec, annealing at 50°C for 30 sec, polymerization at 72°C for 90 sec and final extension at 72°C for 10 minutes. 2.5 pi of amplification product was used for ELISA according to the manufacturer's instructions. The absorbance at 450 nm (with a reference of 690 nm) of each well was measured using Dynex MRX Revelation and Revelation TC 96 Well Microplate Reader.
[0065] Telomerase activity was calculated as suggested in the kit's manual and compared with a control template of 0.1 amol telomeric repeats, representing a relative telomerase activity (RTA) of 100. Inactivated samples and lysis buffer served as negative controls. 2.2. Results
New TERT encoding plasmids are functional in vitro after transfection [0066] The functionality of the new plasmid constructs is shown by the presence of the plasmid encoded TERT protein in the total protein lysate of pCDT or pDUV5 transfected cells in vitro. The inventors performed western-blot assays on the total protein lysate of 293T cells plasmids transfected with pCDT or pDUV5 (24h after transfection). As the TERT protein sequence encoded by each plasmid was tagged with the V5 protein sequence, anti-V5 antibody coupled with Horse Radish Peroxidase (HRP) was used to reveal the presence of the fusion protein of interest.
[0067] A highly positive V5 specific-signal was detected 24 h after transfection in the protein lysate of pCDT or pDUV5 transfected cells. The size of the protein band detected corresponds to the different TERT protein encoded by the plasmids which molecular weight is 123 kDa. Moreover no V5 specific signal was detected in untreated or empty plasmid transfected cells. The inventors demonstrated that pDUV5 and pCDT plasmids were correctly processed in vitro after transfection in mammalian cells and that the plasmid product of expression (TERT protein) was well expressed.
New TERT encoding plasmids express a non-functional enzyme of which cellular expression is excluded from the nucleoli after in vitro transfection [0068] To test the absence of enzymatic activity, a TRAPeze assay was performed. As illustrated by Figure 3, protein lysates from pDUV5 or pCDT transfected cells do not exhibit any telomerase activity. As a positive control, the protein extracts from CrFk cells transfected with the native human TERT (hTERT) were used. Thus the inventors demonstrated that the TERT proteins encoded by either pCDT or pDUV5 plasmids do not express any functional enzymatic activity after in vitro transfection.
[0069] The inventors have further investigated the intracellular location of the two plasmid products of expression. To this aim, an in vitro immunofluorescence assay was performed. Briefly, 24 h after in-vitro transfection of 293T cells with either pCDT or pDUV5, an anti-V5 antibody coupled to an Alexa-Fluor labeled secondary antibody were used to detect the TERT proteins within the cells. The pCDT and pDUV5 encoded TERTs were not detected inside the cell nucleoli contrary to what was observed with 293T cells transfected with the plasmid encoding the native human TERT.
[0070] To conclude, the inventors demonstrated that after in vitro transfection with either pDUV5 and pCDT plasmids, first the TERT protein expression is excluded from the nucleoli and secondly, these products of expression do not exhibit any enzymatic activity. These two criteria establish the safety of the plasmids and favour their use for in vivo vaccination.
Example 3 : In vivo immune response 3.1. Materials and methods
Mice [0071] Female Balb/cBy and C57BL/6J mice (6-8 week old) were purchased from Janvier laboratories (Saint-Berthevin, France). Animals were housed at the Specific Pathogen Free animal facility of the Pasteur Institute. Mice were anesthetized prior to intradermal (ID) or intramuscular (IM) immunizations, with a mix solution of xylazine 2% (Rompun, Bayer Santé, Loos, France) and Ketamine 8% (Imalgen 1000, Merial, Lyon, France) in Phosphate Buffer Saline IX (PBS IX, Life technologies SAS, Saint-Aubin, France), according to individual animal weight and duration of anesthesia (intraperitoneal route). All animals were handled in strict accordance with good animal practice and complied with local animal experimentation and ethics committee guidelines of the Pasteur Institute of Paris. H2 restricted peptides [0072] TERT peptides used in mouse studies (ΙΡΝγ ELIspot) were predicted by in-silico epitope prediction in order to bind mouse class I MHC, H2Kb, H2Db or mouse class II H2-IAd using four algorithms available online:
Syfpeithi (http://www.syfpeithi.de/), Bimas (http://www-bimas.cit.nih.gov/), NetMHCpan and SMM (http://tools.immuneepitope.org/main/).
All synthetic peptides were purchased lyophilized (>90% purity) from Proimmune (Oxford, United Kingdom). Lyophilized peptides were dissolved in sterile water at 2 mg/mL and stored in 35 pL aliquots at -20°C prior use. Details of peptides sequence and H2 restriction is shown in table 1.
Table 1: H2 restricted peptides sequences determined by in silico prediction algorithms
Mice immunization and in vivo electroporation [0073] Intradermal (ID) immunization was performed on the lower part of the flank with Insulin specific needles (U-100, 29GX1/2"-0.33X12 mm, Terumo, Belgium) after shaving. No erythema was observed after shaving, during and after immunization procedure. Intramuscular immunization (IM) was performed in the anterior tibialis cranialis muscle, also using Insulin specific needles U-100. Each animal received a priming dose of either pCDT or pDUV5, independently of vaccine route, corresponding to 100 pg of DNA. All animals were boosted at day 14 post-prime using the same amount of plasmid and the same route of immunization. Directly after ID vaccination, invasive needle electrodes (6X4X2, 47-0050, BTX, USA) are inserted into the skin so that the injection site is placed between the two needle rows (the two needle rows are 0.4 cm apart). Two pulses of different voltages were applied (HV-LV): HV= 1125 V/cm (2 pulses, 50 ps-0.2 ps pulse interval) and LV= 250 V/cm (8 pulses, 100 V-10 ms-20 ms pulse interval). Immediately after IM immunization the muscle injection site was covered with ultrasonic gel (Labo FH, blue contact gel, NM Medical, France) and surrounded by tweezers electrodes (0.5 cm apart, tweezertrode 7 mm, BTXI45-0488, USA) and voltage was applied using the same parameters than for skin electroporation. The Agilepulse® in vivo system electroporator was used for all experiments (BTX, USA).
For each route of immunization (IM, ID) control mice were treated with the same procedures using the same volume of PBS IX. IFNy ELispot assay [0074] Briefly, PVDF microplates (IFN-γ Elispot kit, Diaclone, Abcyss, France, 10 X 96 tests, ref. 862.031.01 OP) were coated overnight with capture antibody (anti-mouse IFN-γ) and blocked with PBS 2% milk. Spleens from pDNA-immunized mice were mashed and cell suspensions were filtered through a 70-mm nylon mesh (Cell Strainer, BD Biosciences, France). Ficoll-purified splenocytes (Lymphocyte Separation Medium, Eurobio, France) were numerated using the +Cellometer® Auto T4 Plus counter (Ozyme, France) and added to the plates in triplicates at 2 χ 105 or 4 χ 105 cells/well and stimulated with 5 pg/ml of dTERT or hyTERT relevant peptides or Concanavalin A (10 pg/ml), or mock stimulated with serum free culture medium. After 19 hours, spots were revealed with the biotin-conjugated detection antibody followed by streptavidin-AP and BCIP/NBT substrate solution. Spots were counted using the Immunospot ELIspot counter and software (CTL, Germany).
Dog TERT peptide pools
[0075] The vast majority of peptides were 15 residues long. A few are 14 amino acids long.
Pool 2 PQKPGAARRMRRLPA, GAARRMRRLPARYWR, RMRRLPARYWRMRPL, LPARYWRMRPLFQEL, YWRMRPEFQELLGNH, RPFFQFLLGNHARCP , QELLGNHARCPYRAL, GNHARCPYRALLRTH, RCPYRALLRTHCPLR, RALLRTHCPLRAMAA, RTHCPLRAMAAKEGS, PLRAMAAKEGSGNQA, MAAKEGSGNQAHRGV, EGSGNQAHRGVGICP, NQAHRGVGICPLERP,
RGVGICPLERPVAAP, ICPLERPVAAPQEQT, PQKPGAARRMRRLPA
Pool 4 AKLSLQELTWKMKVR, LQELTWKMKVRDCTW, TWKMKVRDCTWLHGN, KVRDCTWLHGNPGAC, CTWLHGNPGACCVPA, HGNPGACCVPAAEHR, GACCVPAAEHRRREE, VPAAEHRRREEILAR, EHRRREEILARFLVL, REEILARFLVEVDGH, LARFLVLVDGHIYVV, LVLVDGHTYVVKELR, DGHIYVVKLLRSFFY, YVVKLLRSFFYVTET, LLRSFFYVTETTFQK,
FFYVTETTFQKNRLF, TETTFQKNRLFFYRK, FQKNRLFFYRKSVW
Pool 6 FGGPPGTRPTTPAWH, PGTRPTTPAWHPYPG, PTTPAWHPYPGPQGV, AWHPYPQPQGVPHDP, YPGPQGVPHDPAHPE, QGVPHDPAHPETKRF, HDPAHPETKRFLYCS, HPETKRFLYCSGGRE, KRFLYCSGGRERLRP, YCSGGRERLRPSFLL, GRERLRPSFLLSALP, LRPSFLLSALPPTLS,
FFFSALPPTLSGARK, ALPPTLSGARKLVET
Pool 10 DCTWLHGNPGACCVP, LHGNPGACCVPAAEH, PGACCVPAAEHRRRE, CVPAAEHRRREEIFA, AEHRRREEILARFLV, RREEILARFLVLVDG, ILARFLVLVDGHIYV, FLVLVDGHIYWKLL, VDGHIYWKLLRSFF, IYVVKLLRSFFYVTE, KLLRSFFYVTETTFQ, SFFYVTETTFQKNRL,
VTETTFQKNRLFFYR, TFQKNRLFFYRKSVW
Pool 19 QFPFNQPVRKNPSFF, NQPVRKNPSFFLRVI, RKNPSFFLRVTADTA, SFFLRVIADTASCCY, RVIADTASCCYSLLK, DTASCCYSLLKARNA, CCYSLLKARNAGLSL, LLKARNAGFSLGAKG, RNAGLSLGAKGASGL, LSLGAKGASGLFPSE, AKGASGLFPSEAARW, SGLFPSEAARWFCLH,
PSEAARWLCLHAFL, ARWLCLHAFLLKLAH
In vivo cytotoxicity assay [0076] Briefly, for target cell preparation, splenocytes from naive C57/B16 mice were labeled in PBS IX containing high (5 pM), medium (1 pM) or low (0.2 pM) concentrations of CFSE (Vybrant CFDA-SE cell-tracer kit; Life technologies SAS, Saint-Aubin, France). Splenocytes labeled with 5 and 1 pM CFSE were pulsed with 2 different H2 peptides at 5 pg/ ml for 1 hour and 30 minutes at room temperature. Peptides 987 and 621 were used for pulsing respectively CFSE high and medium labeled naive splenocytes. CFSE low labeled splenocytes were left unpulsed. Each mouse previously immunized with either pCDT or pDUV5 received at day 10 post-boost injection 107 CFSE-labeled cells of a mix containing an equal number of cells from each fraction, through the retro-orbital vein. After 15-18 hours, single-cell suspensions from spleens were analyzed by flow cytometry MACSQUANT® cytometer (Miltenyii, Germany).
The disappearance of peptide-pulsed cells was determined by comparing the ratio of pulsed (high/medium CFSE fuorescence intensity) to unpulsed (low CFSE fuorescence intensity) populations in pDNA immunized mice versus control (PBS IX injected) mice. The percentage of specific killing per test animal was established according to the following calculation: [1 -[mean(CFSE,owPBS/CFSEt’igWmBdiuniPBS)/(CFSElowpDNA/CFSEhigt,/mediunipDNA)]] x 100.
Statistical analysis and data handling [0077] Prism-5 software was used for data handling, analysis and graphic representations. Data are represented as the mean ± standard deviation. For statistical analyses of EliSPOT assays we used a Mann Whitney non parametric test, and a Kruskal-Wallis analysis with Dunn's multiple comparison test for in-vivo cytotoxicity assay. Significance was set at p-value < 0.05. 3.2. Results pDUV5 induces a strong cytotoxic CD8 T cell response after ID or IM immunization and EP in mice [0078] The inventors have assessed whether pDUV5 plasmid DNA plasmid was capable of eliciting efficient cellular immune responses (CD8) in mice. To this aim, different groups of 9-10 C57-B1/6 mice were injected ID or IM with pDUV5 immediately followed by electroporation. Two weeks later, mice received a boost injection with the same protocol. On day 10 post-boost, mice spleens were harvested and the induced immune response was monitored via an IFN-y ELISPOT assay using H2 restricted peptides described in Table 1. Dog TERT peptides restricted to mouse MHC class I were predicted in silico as described in the material and methods section. As shown in Figure 4, a significant augmentation in the frequency of dTERT specific IFN-γ secreting CD8 T-cells was observed in the spleen of ID and IM vaccinated animals in comparison with control mice. This was observed for 2 out of 3 peptides (p621 and p987) (p<0.05). No significant difference was observed between the 2 routes of administration. pDUV5 construct is able to promote the expansion of dTERT specific CD8 T-cells in mice. The inventors next wanted to show that those specific T-cells exhibit a functional cytotoxic activity in vivo, which will be necessary to attack tumor cells. In order to measure the in vivo cytolytic strength of the CD8+ T-cell response elicited by pDUV5 immunization, the inventors performed in vivo cytotoxicity tests using carboxyfuorescein-diacetate suc-cinimidyl ester (CFSE)-labelled, peptide-pulsed splenocytes as target cells. 7 week old C57/B16 mice which received a prime and boost vaccination with pDUV5 via the ID or IM route as described before or mock-immunized with phosphate- buffered saline (PBS) were intravenously injected with 107 target cells. Target cells were splenocytes from naive congenic mice separately labelled with three different concentrations of CFSE and pulsed with individual peptides (p621 or p987) or left unpulsed as an internal control. After 15-18 hours, spleen cells were obtained and the disappearance of peptide-pulsed cells in control versus immunized mice was quantifed by fluorescence-activated cell sorting.
Results show that mice develop CTLs against the 2 epitopes predicted in silico (Figures 5A and 5B). Peptide 621 gives the strongest in vivo lysis. Results were concordant with IFN-y Elispot assays (Figure 4). No significant difference was observed between the two routes of immunization. pCDT induces a strong cytotoxic CD8 T cell response along with a specific CD4 T cell response after ID or IM immunization and electroporation in mice [0079] In light of the importance of cytotoxic CD8 T cells in antitumor immune responses, the inventors have assessed whether plasmid pCDT was able to promote such an immune response in vivo. Thus, different groups of 9-10 C57-B1/6 mice were immunized with pCDT by ID or IM injection of the plasmid immediately followed by electroporation. Two weeks later, mice received a boost injection with the same protocol. On day 10 post-boost, mice spleens were harvested and the induced immune response was monitored via an IFN-γ ELISPOT assay using H2 restricted peptides described in Table 1.
Hy-TERT peptides restricted to mouse MHC class I were predicted in silico as described in the material and methods section. As shown in Figure 6A, a significant augmentation in the frequency of hyTERT specific IFN-γ secreting CD8 T-cells was observed in the spleen of ID and IM vaccinated animals in comparison with control mice. This was observed for 2 out of 3 class I restricted peptides (p621 and p987, p<0.05). No significant difference in the frequency of specific CD8 T cells was observed between IM and ID route for both peptides p921 and p987.
The inventors have further investigated the hyTERT restricted CD4 T cell response. To this aim, 9-10 Balb/C mice were immunized with pCDT by ID or IM injection immediately followed by electroporation and the CD4 specific T cell response was monitored in the spleen as described before using hyTERT IAd restricted peptides (in silico prediction). Balb/C mice were chosen because this mouse strain is known to develop good CD4 T cell responses. As shown in Figure 6B, when performing the IFN-γ ELISPOT assay, a significant augmentation in the frequency of hyTERT specific IFN-γ secreting CD4 T-cells was observed in the spleen of ID and IM vaccinated Balb/C mice in comparison with control mice injected with PBS IX. This was observed for 2 out of 3 class I restricted peptides (p1106 and p1105, with respectively for p1106 p<0.05 for ID route and p<0.001 for IM route and for 1105 the difference was not significant for ID route and p<0.01 for IM route). No significant difference in the frequency of specific CD4 T cells was observed between IM and ID route for both peptides p1105 and p 1106. Thus, pCDT construct is able to promote the expansion of hyTERT specific CD8 and CD4 T-cells in mice. The inventors next wanted to show that hyTERT specific CD8 T-cells exhibit a functional cytotoxic activity in vivo, which will be necessary to destroy tumor cells. In order to measure the in vivo cytolytic strength of the CD8+ T-cell response elicited by pCDT immunization, the inventors performed an in vivo cytotoxicity test using carboxyfluorescein-diacetate succinimidyl ester (CFSE)-labelled, peptide-pulsed splenocytes as target cells. 7 week old C57/B16 mice which received a prime and boost vaccination with pCDT via the ID or IM route as described before or mock-immunized with phosphate-buffered saline (PBS) were intravenously injected with 107 target cells. Target cells were splenocytes from naive congenic mice separately labelled with three different concentrations of CFSE and pulsed with individual peptides (p621 or p987) or left un-pulsed as an internal control. After 15-18 hours, spleen cells were obtained and the disappearance of peptide-pulsed cells in control versus immunized mice was quantified by fluorescence-activated cell sorting.
[0080] Results show that mice develop CTLs against the 2 peptides p621 and p987 which were predicted in silico. Peptide 987 gives the strongest in vivo lysis. Results were consistent with the ones from the IFN-γ Elispot assays (Figure 6A). It is worth mentioning that for p621, the mean percent lysis was slightly superior when pCDT was injected via the ID route (mean ID = 7.7% vs mean IM =0.2%), however, no significant difference was observed between the two routes of immunization.
Example 4 : dog TERT specific T cell repertoire 4.1. Materials and methods
Dog TERT peptides library [0081] Lyophilized dTERT peptides (purity > 90%) were purchased from JPT Peptide Technologies (Berlin, Germany). Each peptide was resuspended in distilled H2O, 5% DMSO at 2 mg/mL prior use according to supplier recommendation and kept frozen at -20°C before use. One third of the dog TERT peptide (AA281 to 571) was used to synthetized 70 peptides of 15 AA overlapping of 11 AAand recovering this sequence of the dog TERT as depicted in Figure 3. Four pools of peptides were used for in vitro experiments and ELIspot assays in dogs.
Canine blood products [0082] Canine blood samples were purchased from the Bourgelat Institute (Marcy I'Etoile, France). It was taken from a healthy 4-year-old beagle dog housed, fed and cared for in accordance with institutional and ethical guidelines. Heparinized blood samples was 4 time diluted in PBS IX (Life technologies SAS, Saint-Aubin, France). Diluted samples were then layered on Lymphocyte Separation Medium (Eurobio, Courtaboeuf, France) and centrifuged 30 minutes at 2200 rpm (at room temperature) without break. Canine PBMCs were harvested and stored in Fetal Calf Serum (FCS, PAA Laboratories GmbH, Pashing, Austria) with 10% DMSO (Sigma Aldrich chimie SARL, Saint-Quentin Fallavier, France) in liquid nitrogen prior use.
In vitro immunization assays in dogs PBMCs [0083] On day 0, dog frozen PBMCs were recovered, counted using the Cellometer® Auto T4
Plus counter (Ozyme, France) and plated in duplicates or triplicates at 106 cells/mL in 48-well flat-bottomed plates (BD, France) in AIM-V medium (Invitrogen) supplemented with either 100 ng/mL caGM-CSF and 5 ng/mL calL-4 (R&amp;DSystems) or 50 ng/mL human FIT3L (Immunotools). Cells were cultured at 37°C, 5% CO2 in an incubator.
[0084] After 24 hours (day1), maturation stimuli were added, comprising the following reagent: 50 ng/mL rcTNFa, 20 ng/mL rclLI-β (R&amp;DSystems), 1 ng/mL hlL-7 (Miltenyi). Pools of peptides were also added. The final concentration used for each peptide was 10 pg/mL. Control wells received the cocktails of maturation cytokines only and no peptide. At day 3, culture medium was discarded and fresh AIM-V was added. Fresh AIM-V was added every 3 days until the day of testing. At either day 11 or day 18 after the beginning of culture, cells were recovered, washed in fresh AIM-V medium and used for the ELIspot assay.
[0085] Briefly cells were plated with the 4 pools of peptides (5 pg/mL of each peptide) in AIMV-5 or in AIMV only. Concanavalin A (10 pg/ml) and recombinant canine IFN-γ (16ng/mL) was used for positive control weels. After 24 hours, spots were revealed with the biotin-conjugated detection antibody followed by streptavidin-AP and BCIP/NBT substrate solution. Spots were counted using the Immunospot ELIspot counter and software (CTL, Germany). 4.2. Results [0086] In order to highlight the relevance of the vaccine technology of the invention, the inventors wanted to demonstrate the existence of a pre-existing dogTERT specific T-cell repertoire in the target species, i.e dogs.
[0087] The inventors have investigated whether dTERT-specificT-cell responses could be enhanced in PBMCs incubated with either rcGM-CSF and rclL-4 for 24 hours, or with hFlt3 ligand followed by maturation stimuli (rclL1 β, rcTNFa and IL-7) and peptides stimulation for another 24 hours and 11 or 18 days of/n vitro cell expansion. This technique was described by Mallone and colleagues for human PBMCs and is called in vitro immunization (Martinuzzi et al. 2011). The principle of this experiment is exposed in Figure 8. To stimulate specific T cells, 15mer overlapping peptides recovering one third of the dog TERT protein (Figure 3) were used in pools containing 17 to 18 peptides each.
[0088] Eleven or 18 days after the beginning of culture, cells were subsequently transferred into dog IFN-γ ELISPOT plates for 24 hours with 5 pg/mL of each pool of peptides. The inventors have noticed a threefold increase in the frequency of dTERT specific IFN-γ secreting T cells with pool 2/rcGMCSF+rclL-4 and a twofold increase with pool 4/FIT-3L after 11 days of culture in comparison with medium stimulated PBMCs (Figure 9A). Moreover, a fourfold increase in the frequency of dTERT specific IFN-γ secreting T cells was observed after 18 days of culture with pool 4/FIT3L in comparison with PBMCs stimulated with culture medium (Figure 9B). These results demonstrate the existence of a naturally occurring repertoire of dog TERT specific IFN-γ secreting T-cell repertoire in peripheral blood of naive experimentation dog.
Example 5 : In vivo specific cellular immune response in dogs vaccinated with pDUV5 [0089] Six naive beagle dogs received a local anaesthetic of 2.5 mg/kg IV imalgene and 20-80 pg/kg IV dorbene 15-20 minutes before vaccination and 100-400 pg/kg IM post vaccination. The dogs were injected intradermally with 400 pg of pDUV5 DNA followed by electroporation. pDU5 DNA was electroporated at days 0, 29, 57 and 142. Peripheral blood was drawn and mononuclear cells tested for dog telomerase specific peptides belonging either to pool 6 or pool 19 according to the method of Martinuzzi et al., 2011.
[0090] Figures 10A and 10B show that ΙΡΝγ specific T cell responses were detected. As shown on Figures 11A and 11B, pDUV5 DNA vaccination at days 57 and 142 show classical long term memory responses, that is rising sharply and decaying more slowly.
Example 6 : Specific dTERT T cell responses in animals with neoplasias [0091] To show that pDUV5 DNA electroporation can induce specific dTERT T cell responses in animals with neoplasias, five pet dogs with neoplasias and three pet dogs as controls were used. The diseased animals presented with widely different tumours. See Table 2 below.
Table 2: Data for healthy and tumor bearing dogs:
[0092] Peripheral blood was drawn and the in vitro stimulation protocol as described in Example 4 and in Martinuzzi zt al.,2011, was performed, using pool 4 peptides.
[0093] As can be seen in Figure 12, specific peptide responses well over medium controls were identified for all animals. This means that the immunological repertoire is not depleted, biased or suppressed by the neoplasias. The latter finding is particularly important for it shows that even if there was some degree of immunosuppression or excessive Treg induction in diseased dogs, the vaccination of the invention is nonetheless capable of inducing T cell responses.
References [0094]
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SEQUENCE LISTING
[0095] <110> intelectys <120> A cancer vaccine in dogs
<130> B1581PC <160> 14 <170> Patentln version 3.3 <210> 1 <211> 3567
<212> DNA <213> Artificial <220> <223> pDUV5 plasmid sequence <220>
<221 > CDS <222> (13)..(3555) <400> 1 ggatccgccg cc atg cag att ttc gtc aaa acc ctc acc ggc aag acc atc 51
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Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys 15 20 25 atc cag gat aag gag ggc att cct cct gac cag cag aga ett att ttc 147
Ile Gin Asp Lys Glu Gly Ile Pro Pro Asp Gin Gin Arg Leu Ile Phe 30 35 40 45 gea ggc aaa cag ctg gag gac ggc aga aca ttg tet gac tae aac atc 195
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Glu Thr Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val 160 165 170 ggc gac gat gtg ttg aca cac ctg etc gcc agg tgc gca ctt tac ctg 579
Gly Asp Asp Val Leu Thr His Leu Leu Ala Arg Cys Ala Leu Tyr Leu 175 180 185 ctg gtg gcc cca agt tgc gcc tac cag gtg tgc gga cct cct ttg tac 627
Leu Val Ala Pro Ser Cys Ala Tyr Gin Val Cys Gly Pro Pro Leu Tyr 190 195 200 205 gac etc tgt gcc cct gcc tct ttg cca ctg cct gcc cct ggc ctg cct 675
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Ala Gin Gin Pro Pro Val Ser Glu Ala Pro Ala Val Thr Pro Ala Val 290 295 300 gcc gee age cct gee gee tea tgg gaa gga gga ccc cct gga acc agg 963
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Pro Thr Thr Pro Ala Trp His Pro Tyr Pro Gly Pro Gin Gly Val Pro 320 325 330 cat gat cct get cac cca gaa acc aag egg ttc ctg tac tgc age gga 1059
His Asp Pro Ala His Pro Glu Thr Lys Arg Phe Leu Tyr Cys Ser Gly 335 340 345 ggt aga gaa cgc ttg cgc cca age ttt ctg etc age gcc ctg cct cca 1107
Gly Arg Glu Arg Leu Arg Pro Ser phe Leu Leu Ser Ala Leu Pro Pro 350 355 360 365 act ctt tec gga gcc egg aaa etc gtg gaa acc atc ttt etc ggt age 1155
Thr Leu Ser Gly Ala Arg Lys Leu Val Glu Thr lie Phe Leu Gly Ser 370 375 380 get cct cag aaa cca gga gcc get agg egg atg cgc aga ctg cct gca 1203
Ala Pro Gin Lys Pro Gly Ala Ala Arg Arg Met Arg Arg Leu Pro Ala 385 390 395 cgc tac tgg cgc atg cgc cca etc ttt cag gag ctg ctg gga aat cat 1251
Arg Tyr Trp Arg Met Arg Pro Leu Phe Gin Glu Leu Leu Gly Asn His 400 405 410 gca agg tgc ccc tat egg get ctg ctt egg act cac tgt cca ctg aga 1299
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Gly lie Cys Pro Leu Glu Arg Pro Val Ala Ala Pro Gin Glu Gin Thr 450 455 460 gat tea acc cgc ett gtg cag ctg etc agg cag cat agt tee cct tgg 1443
Asp Ser Thr Arg Leu Val Gin Leu Leu Arg Gin His Set Ser Pro Trp 465 470 475 cag gtg tat gca ttc ctg aga get tgc ctg tgc tgg ctg gtg cca acc 1491
Gin Val Tyr Ala Phe Leu Arg Ala Cys Leu Cys Trp Leu Val Pro Thr 480 485 490 ggc etc tgg ggc agt aga cac aac cag agg cgc ttt ctg egg aac gtg 1539
Gly Leu Trp Gly Ser Arg His Asn Gin Arg Arg Phe Leu Arg Asn val 495 500 505 aaa aag ttt atc tet etc gga aaa cac get aag ctg age etc cag gaa 1587
Lys Lys Phe Tie Ser Leu Gly Lys His Ala Lys Leu Ser Leu Gin Glu 510 515 520 525 ctg acc tgg aag atg aag gtg egg gat tgt act tgg etc cac ggc aac 1635
Leu Thr Trp Lys Met Lys Val Arg Asp Cys Thr Trp Leu His Gly Asn 530 535 540 cca ggc get tgc tgc gtt cca get gca gag cac agg agg egg gaa gaa 1683
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His Val Ser Thr Phe Ala Asp Leu Gin Pro Tyr Met Arg Gin Phe Val 785 790 795 gag agg ett cag gaa aca age ctg ett agg gac gca gtg gtg atc gag 2451
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Val Glu Asp Gly Ala Leu Gly Ser Ala Ala Pro Leu Gin Leu Pro Ala 930 935 940 cat tgc ett ttc cct tgg tgt ggc ctg ctg ctg gat acc aga aca ctg 2883
His Cys Leu Phe Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu 945 950 955 gaa gtc tet tgc gat tat tet tcc tat get cac acc agt att egg gee 2931
Glu Val Ser Cys Asp Tyr Ser Ser Tyr Ala His Thr Ser lie Arg Ala 960 965 970 agt ttg act ttt tea cag ggc get aaa cca gga cgc aat atg aga egg 2979
Ser Leu Thr Phe Ser Gin Gly Ala Lys Pro Gly Arg Asn Met Arg Arg 975 980 985 aaa ett ctg gee gtt ttg egg ctg aaa tgc tgt gee ctg ttc ctg gat 3027
Lys Leu Leu Ala Val Leu Arg Leu Lys Cys Cys Ala Leu Phe Leu Asp 990 995 1000 1005 ctg cag gtc aat ggc att cat acc gtt tat atg aac gtc tat aag 3072
Leu Gin Val Asn Gly He His Thr Val Tyr Met Asn Val Tyr Lys 1010 1015 1020 ate ttc ctg ett cag gcc tac aga ttt cac get tgc gtg ctg cag 3117
He Phe Leu Leu Gin Ala Tyr Arg Phe His Ala Cys Val Leu Gin 1025 1030 1035 ctg ccc ttc aat cag ccc gtg egg aaa aac ccc age ttc ttt ctt 3162 Léu Pro Phe Asn Gin Pro val Arg Lys Asn Pro Ser phe Phe Leu 1040 1045 1050 ego gtc ate gca gat aca gca tcc tgt tgc tat tcc ttg ctt aag 3207
Arg Val lie Ala Asp Thr Ala Ser Cys Cys Tyr Ser Leu Leu Lys 1055 1060 1065 gca aga aat get gga ctg tea etc ggt get aag ggt gcc age ggc 3252
Ala Arg Asn Ala Gly Leu Ser Leu Gly Ala Lys Gly Ala Ser Gly 1070 1075 1080 ttg ttt cca age gag get gcc agg tgg ttg tgt ctt cac gca ttc 3297
Leu Phe Pro Ser Glu Ala Ala Arg Trp Leu Cys Leu His Ala Phe 1085 1090 1095 ttg ctg aaa ttg get cac cat age ggc aca tat agg tgt ctg ctg 3342
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<212> PRT <213> Artificial <220> <223> Synthetic Construct <400>2
Met Gin lie Phe Val Lys Thr Leu Thr Gly Lys Thr lie Thr Leu Glu 1 5 10 15
Val Glu Pro Ser Asp Thr lie Glu Asn Val Lys Ala Lys lie Gin Asp 20 25 30
Lys Glu Gly lie Pro Pro Asp Gin Gin Arg Leu lie Phe Ala Gly Lys 35 40 45
Gin Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn lie Gin Lys Glu 50 55 60
Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Arg Ala Leu Val 65 70 75 80
Ala Gin Cys Leu Val Cys Val Pro Trp Gly Ala Arg Pro Pro Pro Ala 85 90 95
Ala Pro Cys Phe Arg Gin Val Ser Cys Leu Lys Glu Leu Val Ala Arg 100 105 110
Val Val Gin Arg Leu Cys Glu Arg Gly Ala Arg Asn Val Leu Ala Phe 115 120 125
Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro Val Ala Phe 130 135 140
Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr Glu Thr Leu 145 150 155 160
Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val Gly Asp Asp 165 170 175
Val Leu Thr His Leu Léu Ala Arg Cys Ala Leu Tyr Leu Leu Val Ala 180 185 190
Pro Ser Cys Ala Tyr Gin Val Cys Gly Pro Pro Leu Tyr Asp Leu Cys 195 200 205
Ala Pro Ala Ser Leu Pro Leu Pro Ala Pro Gly Leu Pro Gly Leu Pro 210 215 220
Gly Leu Pro Gly Leu Gly Ala Gly Ala Gly Ala Ser Ala Asp Leu Arg 225 230 235 240
Pro Thr Arg Gin Ala Gin Asn Ser Gly Ala Arg Arg Arg Arg Gly Ser 245 250 255
Pro Gly Ser Gly Val Pro Leu Ala Lys Arg Pro Arg Arg Ser Val Ala 260 265 270
Ser Glu Pro Glu Arg Gly Ala His Arg Ser Phe Pro Arg Ala Gin Gin 275 280 285
Pro Pro Val Ser Glu Ala Pro Ala Val Thr Pro Ala Val Ala Ala Ser 290 295 300
Pro Ala Ala Ser Trp Glu Gly Gly Pro Pro Gly Thr Arg Pro Thr Thr 305 310 315 320
Pro Ala Trp His Pro Tyr Pro Gly Pro Gin Gly Val Pro His Asp Pro 325 330 335
Ala His Pro Glu Thr Lys Arg Phe Leu Tyr Cys Ser Gly Gly Arg Glu 340 345 350
Arg Leu Arg Pro Ser Phe Leu Leu Ser Ala Leu Pro Pro Thr Leu Ser 355 360 365
Gly Ala Arg Lys Leu Val Glu Thr lie Phe Leu Gly Ser Ala Pro Gin 370 375 380
Lys Pro Gly Ala Ala Arg Arg Met Arg Arg Leu Pro Ala Arg Tyr Trp 385 390 395 400
Arg Met Arg Pro Leu Phe Gin Glu Leu Leu Gly Asn His Ala Arg Cys 405 410 415
Pro Tyr Arg Ala Leu Leu Arg Thr His Cys Pro Leu Arg Ala Met Ala 420 425 430
Ala Lys Glu Gly Ser Gly Asn Gin Ala His Arg Gly Val Gly He Cys 435 440 445
Pro Leu Glu Arg Pro Val Ala Ala Pro Gin Glu Gin Thr Asp Ser Thr 450 455 460
Arg Leu Val Gin Leu Leu Arg Gin His Ser Ser Pro Trp Gin Val Tyr 465 470 475 480
Ala Phe Leu Arg Ala Cys Leu Cys Trp Leu Val Pro Thr Gly Leu Trp 485 490 495
Gly Ser Arg His Asn Gin Arg Arg Phe Leu Arg Asn Val Lys Lys Phe 500 505 510
He Ser Leu Gly Lys His Ala Lys Leu Ser Leu Gin Glu Leu Thr Trp 515 520 525
Lys Met Lys Val Arg Asp Cys Thr Trp Leu His Gly Asn Pro Gly Ala 530 535 540
Cys Cys Val Pro Ala Ala Glu His Arg Arg Arg Glu Glu He Leu Ala 545 550 555 560
Arg Phe Leu Val Leu Val Asp Gly His He Tyr Val Val Lys Leu Leu 565 570 575
Arg Ser Phe Phe Tyr Val Thr Glu Thr Thr Phe Gin Lys Asn Arg Leu 580 585 590
Phe Phe Tyr Arg Lys Ser Val Trp Ser Gin Leu Gin Ser He Gly lie 595 600 605
Arg Gin Leu Phe Asn Ser Val His Leu Arg Glu Leu Ser Glu Ala Glu 610 615 620
Val Arg Arg His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu 625 630 635 640
Arg Phe Leu Pro Lys Pro Ser Gly Leu Arg Pro He Val Asn Met Asp 645 650 655
Tyr He Met Gly Ala Arg Thr Phe His Arg Asp Lys Lys Val Gin His 660 665 670 τ rrvu«. /’’Ί»» T «... t..„ rrru.» t x.« nk« τ /'I.. * xi^u xiix oex <jxxi xj«u xjys xxxx j-i«u rne otsx vax xi«su λ»ιι xyx i?xu Aiy 675 680 685
Ala Arg Arg Pro Ser Leu Leu Gly Ala Ser Met Leu Gly Met Asp Asp 690 695 700
Ile His Arg Ala Trp Arg Thr Phe Val Leu Arg Ile Arg Ala Gin Asn 705 710 715 720
Pro Ala Pro Gin Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr 725 730 735
Asp Ala Leu Pro Gin Asp Arg Leu val Glu val ile Ala Asn val ile 740 745 750
Arg Pro Gin Glu Ser Thr Tyr Cys Val Arg His Tyr Ala Val Val Gin 755 760 765
Arg Thr Ala Arg Gly His Val Arg Lys Ala Phe Lys Arg His Val Ser 770 775 780
Thr Phe Ala Asp Leu Gin Pro Tyr Met Arg Gin Phe Val Glu Arg Leu 785 790 795 800
Gin Glu Thr Ser Leu Leu Arg Asp Ala Val Val Ile Glu Gin Ser Ser 805 810 815
Ser Leu Asn Glu Ala Gly Ser Ser Leu Phe His Leu Phe Leu Arg Leu 820 825 830
Val His Asn His Val Val Arg Ile Gly Gly Lys Ser Tyr Ile Gin Cys 835 840 845
Gin Gly Val Pro Gin Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu 850 855 860
Cys Tyr Gly Asp Met Glu Arg Arg Leu Phe Pro Gly Ile Glu Gin Asp 865 870 875 880
Gly Val Leu Leu Arg Leu Phe Leu Leu Val Thr Pro His Leu Thr Gin 885 890 895
Ala Gin Ala Phe Leu Arg Thr Leu Val Lys Gly Val Pro Glu Tyr Gly 900 905 910
Cys Arg Ala Asn Leu Gin Lys Thr Ala Val Asn Phe Pro Val Glu Asp 915 920 925
Gly Ala Leu Gly Ser Ala Ala Pro Leu Gin Leu Pro Ala His Cys Leu 930 935 940
Phe Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Ser 945 950 955 960
Cys Asp Tyr Ser Ser Tyr Ala His Thr Ser Ile Arg Ala Ser Leu Thr 965 970 975
Phe Ser Gin Giv Ala Lvs Pro Giv Aro Asn Met Aro Aro T.vs Leu Leu 980 " 985 = ” 990
Ala Val Léu Arg Leu Lys Gys Gys Ala Leu Phe Leu Asp Leu Gin Val 995 1000 1005
Asn Gly Ile His Thr Val Tyr Met Asn Val Tyr Lys Ile Phe Leu 1010 1015 1020
Leu Gin Ala Tyr Arg Phe His Ala Cys Val Leu Gin Leu Pro Phe 1025 1030 1035
Asn Gin Pro Val Arg Lys Asn Pro Ser Phe Phé Leu Arg Val Ile 1040 1045 1050
Ala Asp Thr Ala Ser Cys Cys Tyr Ser Leu Leu Lys Ala Arg Asn 1055 1060 1065
Ala Gly Leu Ser LeU Gly Ala Lys Gly Ala Ser Gly Leu Phe Pro 1070 1075 1080
Ser Glu Ala Ala Arg Trp Leu Cys Leu His Ala Phe Leu Leu Lys 1085 1090 1095
Leu Ala His His Ser Gly Thr Tyr Arg Cys Leu Leu Gly Ala Leu 1100 1105 1110
Gin Ala Ala Lys Ala His Leu Ser Arg Gin Leu Pro Arg Gly Thr 1115 1120 1125
Leu Ala Ala Leu Glu Ala Ala Ala Asp Pro Ser Leu Thr Ala Asp 1130 1135 1140
Phe Lys Thr Ile Leu Asp Thr Glu Leu Lys Leu Ser Asp Tyr Glu 1145 1150 1155
Gly Arg Leu Ile Gin Asn Ser Leu Thr Gly Lys Pro Ile Pro Asn 1160 1165 1170
Pro Leu Leu Gly Leu Asp Ser Thr 1175 1180 <210> 3 <211> 3564
<212> DNA <213> Artificial <220> <223> pCDT plasmid sequence <220>
<221 > CDS <222> (13)..(3552) <400>3 aagcttgccg cc atg cag att ttc gtc aaa acc ctc acc ggc aag acc atc 51
Met Gin Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile 15 10 aca ttg gaa gtg gaa ccc agt gat act atc gaa aat gtt aaa gcc aaa 99
Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys 15 20 25 atc cag gat aag gag ggc att cct cct gac cag cag aga ett att ttc 147
Ile Gin Asp Lys Glu Gly Ile Pro Pro Asp Gin Gin Arg Leu Ile Phe 30 35 40 45 gca ggc aaa cag ctg gag gac ggc aga aca ttg tet gac tac aac atc 195
Ala Gly Lys Gin Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile 50 55 60 cag aaa gag age aca ett cac ttg gtt ctc cgc ett cgc gga gga egg 243
Gin Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Arg 65 70 75 gcc etc gtg get cag tgt ctg gtg tgt gtc cca tgg gga gca egg cct 291
Ala Leu Val Ala Gin Cys Leu Val Cys Val Pro Trp Gly Ala Arg Pro 80 85 90 cca cca gca gcc ccc tgc ttt aga cag gtc agt tgc ctc aag gag ctc 339
Pro Pro Ala Ala Pro Cys Phe Arg Gin Val Ser Cys Leu Lys Glu Leu 95 100 105 gtg gcc agg gtg gtt cag aga ctc tgc gag egg ggt gcc egg aac gtc 387
Val Ala Arg Val Val Gin Arg Leu Cys Glu Arg Gly Ala Arg Asn Val 110 115 120 125 etc get ttt gga ttc gca ctg ctg gac ggc get cgc gga ggc cca ccc 435
Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro 130 135 140 gtg gcc ttt aca acc age gtg egg tea tac ctg ccc aac act gtg aca 483
Val Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr 145 150 155 gag aca ctg aga ggc tee ggc get tgg ggc ett ctg ttg agg cgc gtt 531
Glu Thr Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val 160 165 170 ggc gac gat gtg ttg aca cac ctg ctc gcc agg tgc gca ett tac ctg 579
Gly Asp Asp Val Leu Thr His Leu Leu Ala Arg Cys Ala Leu Tyr Leu 175 180 185 ctg gtg gcc cca agt tgc gcc tac cag gtg tgc gga cct cct ttg tac 627
Leu Val Ala Pro Ser Cys Ala Tyr Gin Val Cys Gly Pro Pro Leu Tyr 190 195 200 205 gac ctc tgt gcc cct gcc tet ttg cca ctg cct gcc cct ggc ctg cct 675
Asp Leu Cys Ala Pro Ala Ser Leu Pro Leu Pro Ala Pro Gly Leu Pro 210 215 220 gga ett cct ggt ctg cct ggt etc ggc get gga get ggc gcc tee gca 723
Gly Leu Pro Gly Leu Pro Gly Leu Gly Ala Gly Ala Gly Ala Ser Ala 225 230 235 gat ctc agg cct acc cgc cag gca cag aat age gga gcc agg cgc cgc 771
Asp Leu Arg Pro Thr Arg Gin Ala Gin Asn Ser Gly Ala Arg Arg Arg 240 245 250 egg ggt age cca ggt tet ggc gtc ccc ctg get aaa aga cca egg agg 819
Arg Gly Ser Pro Gly Ser Gly Val Pro Leu Ala Lys Arg Pro Arg Arg 255 260 265 tea gtt get tee gaa ccc gag egg ggc gca cat cgc tee ttt ccc aga 867
Ser Val Ala Ser Glu Pro Glu Arg Gly Ala His Arg Ser Phe Pro Arg 270 275 280 285 gcc cag cag cca cct gtg tct gag get cca gca gtg aca ccc get gtg 915
Ala Gin Gin Pro Pro Val Ser Glu Ala Pro Ala Val Thr Pro Ala Val 290 295 300 gcc gcc age cct gcc gcc tea tgg gaa gga gga ccc cct gga acc agg 963
Ala Ala Ser Pro Ala Ala Ser Trp Glu Gly Gly Pro Pro Gly Thr Arg 305 310 315 ccc act acc ccc get tgg cac ccc tac cct gga ccc cag ggc gtc cct 1011
Pro Thr Thr Pro Ala Trp His Pro Tyr Pro Gly Pro Gin Gly Val Pro 320 325 330 cat gat cct get cac cca gaa acc aag egg ttc ctg tac tgc age gga 1059
His Asp Pro Ala His Pro Glu Thr Lys Arg Phe Leu Tyr Cys Ser Gly 335 340 345 ggt aga gaa cgc ttg cgc cca agt ttt ctg etc age gee ctg cct cca 1107
Gly Arg Glu Arg Leu Arg Pro Ser Phe Leu Leu Ser Ala Leu Pro Pro 350 355 360 365 act ctt tec gga gcc egg aaa etc gtg gaa acc atc ttt etc ggt age 1155
Thr Leu Ser Gly Ala Arg Lys Leu Val Glu Thr He Phe Leu Gly Ser 370 375 380 get act cag aaa cca gga gcc get agg egg atg cgc aga ctg cct gca 1203
Ala Pro Gin Lys Pro Gly Ala Ala Arc Arc Met Arg Arg Leu Pro Ala 385 390 395 cgc tac tgg cgc atg cgc cca etc ttt cag gag ctg ctg gga aat cat 1251
Arg Tyr Trp Arg Met Arg Pro Leu Phe Gin Glu Leu Leu Gly Asn His 400 405 410 gca agg tgc ccc tat egg get ctg ctt egg act cac tgt cca ctg aga 1299
Ala Arg Cys Pro Tyr Arg Ala Leu Léu Arg Thr His Cys Pro Leu Arg 415 420 425 get atg gca gca aag gaa gga agt gga aac cag gcc cat aga gga gtc 1347
Ala Met Ala Ala Lys Glu Gly Ser Gly Asn Gin Ala His Arg Gly Val 430 435 440 445 ggt atc tgt cca ctg gag cgc ccc gtt get gee ccc cag gaa cag acc 1395
Gly lie Cys Pro Leu Glu Arg Pro Val Ala Ala Pro Gin Glu Gin Thr 450 455 460 gat tea acc cgc ctt gtg cag etc ctg agg cag cac agt age cca tgg 1443
Asp Ser Thr Arg Leu Val Gin Leu Leu Arg Gin His Ser Ser Pro Trp 465 470 475 cag gtg tat get ttt ctt cgc get tgt ctg tgc cgc etc gtg ccc gee 1491
Gin Val Tyr Ala Phe Leu Arg Ala Cys Leu Cys Arg Leu Val Pro Ala 480 485 490 ggt ctg tgg ggc age ggc cac aac aga aga cgc ttt ttg egg aat gtg 1539
Gly Leu Trp Gly Ser Gly His Asn Arg Arg Arg Phe Leu Arg Asn Val 495 500 505 aaa aag ttc gtg tec ctg gga aag cac get aaa ctg tea ttg cag gag 1587
Lys Lys Phe Val Ser Leu Gly Lys His Ala Lys Leu Ser Leu Gin Glu 510 515 520 525 ctg acc tgg aag atg egg gtg cag gat tgt gca tgg ctg agg ggc tct 1635
Leu Thr Trp Lys Met Arg Val Gin Asp Cys Ala Trp Leu Arg Gly Ser 530 535 540 ccc gga gcc cgc tgc gtc cca gcc gcc gaa cac aga egg cgc gag gag 1683
Pro Gly Ala Arg Cys Val Pro Ala Ala Glu His Arg Arg Arg Glu Glu 545 550 555 gtg etc gca aag etc ttg tgc tgg ctg atg gga acc tac gtg gtc gaa 1731
Val Leu Ala Lys Leu Leu Cys Trp Leu Met Gly Thr Tyr Val Val Glu 560 565 570 ctg ctg aaa tct ttt ttc tat gtc act gag act aca ttc cag aag aat 1779
Leu Leu Lys Ser Phe Phe Tyr Val Thr Glu Thr Thr Phe Gin Lys Asn
rtr Ran RSR cgc ctg ttc ttt tac egg aaa agg atc tgg tec cag ett cag age att 1827
Arg Leu Phe Phe Tyr Arg Lys Arg lie Trp Ser Gin Leu Gin Ser He 590 595 600 605 ggc atc egg cag cat ttt aac tet gtt cac ctg agg gag ctg age gag 1875
Gly He Arg Gin His Phe Asn Ser val His Leu Arg Glu Leu Ser Glu 610 615 620 gca gaa gtg agg cgc cat cag gag gcc cgc ccc act ctg ett acc tee 1923
Ala Glu Val Arg Arg His Gin Glu Ala Arg Pro Thr Leu Leu Thr Ser 625 630 635 aag ctg egg ttc ctg cct aaa cca tea ggt ctg aga ccc att gtc aac 1971
Lys Leu Arg Phe Leu Pro Lys Pro Ser Gly Leu Arg Pro He Val Asn 640 645 650 atg gat tac gtg gtg ggc gcc aga aca ttc aga aga gac aaa aag gtt 2019
Met Asp Tyr Val Val Gly Ala Arg Thr Phe Arg Arg Asp Lys Lys Val 655 660 665 egg cat etc acc tea cag gtt aaa aac ctg ttt tet gtt ctg aac tac 2067
Arg His Leu Thr Ser Gin Val Lys Asn Leu Phe Ser Val Leu Asn Tyr 670 675 680 685 gaa agg gcc agg agg cca tea ctg ctg ggt gcc agt gtg ctg gga atg 2115
Glu Arg Ala Arg Arg Pro Ser Leu Leu Gly Ala Ser Val Leu Gly Met 690 695 700 gac gat att cac aga gtc tgg egg age ttc gtg ett egg gtg aga get 2163
Asp Asp lie His Arg Val Trp Arg Ser Phe Val Leu Arg Val Arg Ala 705 710 715 cag gac ccc gcc cca cag ttg tat ttt gtc aag gtc gat gtg act ggt 2211
Gin Asp Pro Ala Pro Gin Leu Tyr Phe Val Lys Val Asp Val Thr Gly 720 725 730 get tat gac get etc cct cag gac aaa ttg gtg gag gtg atc get aat 2259
Ala Tyr Asp Ala Leu Pro Gin Asp LyS Leu Val Glu: Val He Ala Asn 735 740 745 gtc atc cgc ccc cag gaa aat aca tac tgc gtg egg cat tac get gtg 2307
Val lie Arg Pro Gin Glu Asn Thr Tyr Cys Val Arg His Tyr Ala Val 750 755 760 765 gtg cag cgc acc gca cag ggc cac gtg agg aaa tec ttc aag egg cat 2355
Val Gin Arg Thr Ala Gin Gly His Val Arg Lys Ser Phe Lys Arg His 770 775 780 gtg tec acc ttc gtc gac etc cag cca tat atg cgc cag ttt gtg gag 2403
Val Ser Thr Phe Val Asp Leu Gin Pro Tyr Met Arg Gin Phe Val Glu 785 790 795 cac ctg cag gaa act tea age ett agg gat gcc gtt gtt atc gag cag 2451
His Leu Gin Glu Thr Ser Ser Leu Arg Asp Ala Val Val He Glu Gin 800 805 810 agt tet agt etc aac gag acc gga cac agt etc ttc cac etc ttt ctg 2499
Ser Ser Ser Leu Asn Glu Thr Gly His Ser Leu Phe His Leu Phe Leu 815 820 825 agg etc gtg cat aat cat gtc atc cgc att gga gga aaa tet tat gtt 2547
Arg Leu Val His Asn His Val lie Arg He Gly Gly Lys Ser Tyr Val 830 835 840 845 cag tgc cag ggc atc cct cag ggt tet ate ctg tea act ctg etc tgc 2595
Gin Cys Gin Gly lie Pro Gin Gly Ser He Leu Ser Thr Leu Leu Cys 850 855 860 tec ttg tgt tac ggc gat atg gaa agt agg ett ttc tea gga atc cag 2643
Ser Leu Cys Tyr Gly Asp Met Glu Ser Arg Leu Phe Ser Gly lie Gin 865 870 875 cag gac ggc gtc ctg ctg egg ctg ttt ett ctg gtg aca cct cac ctg 2691
Gin Asp Gly Val Leu Leu Arg Leu Phe Leu Leu Val Thr Pro His Leu 880 885 890 gca cag gcc cag gcc ttc ctg cgc aca ctg gtg age gga gtg cct gag 2739
Ala Gin Ala Gin Ala Phe Leu Arg Thr Leu Val Ser Gly Val Pro Glu 895 900 905 tac ggc tgt acc gcc aac ctg cag aag aca gcc gtg aat ttt cca gtg 2787
Tyr Gly Cys Thr Ala Asn Leu Gin Lys Thr Ala Val Asn Phe Pro Val 910 915 920 925 gac acc ggt get cca ggc tcc gcc gca cct ctg cag ttg ccc gca cat 2835
Asp Thr Gly Ala Pro Gly Ser Ala Ala Pro Leu Gin Leu Pro Ala His 930 935 940 tgt etc ttt cct tgg tgt ggc Ctg etc etc gac acc egg act ttg gaa 2883
Cys Leu Phe Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu 945 950 955 gtc ttt tgc gat tac tcc age tat gca cag aca tcc att agg age age 2931
Val Phe Cys Asp Tyr Ser Ser Tyr Ala Gin Thr Ser He Arg Ser Ser 960 965 970 ctg aca ttc age cag ggc aca egg ccc ggc cgc aat atg agg aga aag 2979
Leu Thr Phe Ser Gin Gly Thr Arg Pro Gly Arg Asn Met Arg Arg Lys 975 980 985 ttg etc gee gtt atg aga etc aag tgc tgt gca gtc ttt ett gat ctg 3027
Leu Leu Ala Val Met Arg Leu Lys Cys Cys Ala Val Phe Leu Asp Leu 990 995 1000 1005 cag gtc aat tet att cat acc gtt tac acc aac atc tat aaa att 3072
Gin Val Asn Ser lie His Thr Val Tyr Thr Asn He Tyr Lys He 1010 1015 1020 ttc ctg etc cag gca tat aga ttt cac gcc tgc gtg ttg cag ttc 3117
Phe Leu Leu Gin Ala Tyr Arg Phe His Ala Cys Val Leu Gin Phe 1025 1030 1035 cca ttc aat cag ccc gtt egg aag aac ccc agt ttc ttt etc agg 3162
Pro Phe Asn Gin Pro Val Arg Lys Asn Pro Ser Phe Phe Leu Arg 1040 1045 1050 gtt att get gat acc gcc tcc cgc tgt tac tcc ctg ett aag gcc 3207
Val He Ala Asp Thr Ala Ser Arg Cys Tyr Ser Leu Leu Lys Ala 1055 1060 1065 aag aac aca gga ett tea ttg ggt get aaa ggc gcc agt gga cct 3252
Lys Asn Thr Gly Leu Ser Leu Gly Ala Lys Gly Ala Ser Gly Pro 1070 1075 1080 ttc cct tet gaa gcc get egg tgg etc tgt ttg cac gca ttc ett 3297
Phe Pro Ser Glu Ala Ala Arg Trp Leu Cys Leu His Ala Phe Leu 1085 1090 1095 ctg aag ttg get aga cac age tet act tac aga tgc ett ctg ggc 3342
Leu Lys Leu Ala Arg His Ser Ser Thr Tyr Arg Cys Leu Leu Gly 1100 1105 1110 ccc ett aga get get aag get cat ctg tea aga cag etc cca aga 3387
Pro Leu Arg Ala Ala Lys Ala His Leu Ser Arg Gin Leu Pro Arg 1115 1120 1125 ggc act etc gcc gca ctg gag gcc gca gcc gac ccc tcc etc act 3432
Gly Thr Leu Ala Ala Leu Glu Ala Ala Ala Asp Pro Ser Leu Thr 1130 1135 1140 gca gat ttt aag act att etc gat acc gag ett aag ttg tea gac 3477
Ala Asp Phe Lys Thr lie Leu Asp Thr Glu Leu Lys Leu Ser Asp 1145 1150 1155 tac gag gga cgc ctg att cag aat age ctg aca ggc aaa ccc att 3522
Tyr Glu Gly Arg Leu He Gin Asn Ser Leu Thr Gly Lys Pro He 1160 1165 1170 r’r'l· ΛΛ+ pw* r’-t-rr f-4-rr rrrr+ -t-Frr rra+ 4-r*r» ΛηΛ rra 8^64 *-'**W. WWW WWW ww^ ’tf’tf'- '-'-’tf ’tfW’W WWW WWW WW, MWMM.WW.WM yw .WWW,-».
Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr 1175 1180 <210>4 <211 > 1180
<212> PRT <213> Artificial <220> <223> Synthetic Construct <400>4
Met Gin Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 15 10 15
Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gin Asp 20 25 30
Lys Glu Gly Ile Pro Pro Asp Gin Gin Arg Leu Ile Phe Ala Gly Lys 35 40 45
Gin Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gin Lys Glu 50 55 60
Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Arg Ala Leu Val 65 70 75 80
Ala Gin Cys Leu Val Cys Val Pro Trp Gly Ala Arg Pro Pro Pro Ala 85 90 95
Ala Pro Cys Phe Arg Gin Val Ser Cys Leu Lys Glu Leu Val Ala Arg 100 105 110
Val Val Gin Arg Leu Cys Glu Arg Gly Ala Arg Asn Val Leu Ala Phe 115 120 125
Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro Val Ala Phe 130 135 140
Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr Glu Thr Leu 145 150 155 160
Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val Gly Asp Asp 165 170 175
Val Leu Thr His Leu Leu Ala Arg Cys Ala Leu Tyr Leu Leu Val Ala 180 185 190
Pro Ser Cys Ala Tyr Gin Val Cys Gly Pro Pro Leu Tyr Asp Leu Cys 195 200 205
Ala Pro Ala Ser Leu Pro Leu Pro Ala Pro Gly Leu Pro Gly Leu Pro 210 215 220
Gly Leu Pro Gly Leu Gly Ala Gly Ala Gly Ala Ser Ala Asp Leu Arg 225 230 235 240
Pro Thr Arg Gin Ala Gin Asn Ser Gly Ala Arg Arg Arg Arg Gly Ser 245 250 255
Pro Gly Sér Gly Val Pro Leu Ala Lys Arg Pro Arg Arg Ser Val Ala 260 265 270
Ser Glu Pro Glu Arg Gly Ala His Arg Ser Phe Pro Arg Ala Gin Gin 275 280 285
Pro Pro Val Ser Glu Ala Pro Ala Val Thr Pro Ala Val Ala Ala Ser 290 295 300
Pro Ala Ala Ser Trp Glu Gly Gly Pro Pro Gly Thr Arg Pro Thr Thr 305 310 315 320
Pro Ala Trp His Pro Tyr Pro Gly Pro Gin Gly Val Pro His Asp Pro 325 330 335
Ala His Pro Glu Thr Lys Arg Phe Leu Tyr Cys Ser Gly Gly Arg Glu 340 345 350
Arg Leu Arg Pro Ser Phe Leu Leu Ser Ala Leu Pro Pro Thr Leu Ser 355 360 365
Gly Ala Arg Lys Leu Val Glu Thr lie Phe Leu Gly Ser Ala Pro Gin 370 375 380
Lys Pro Gly Ala Ala Arg Arg Met Arg Arg Leu Pro Ala Arg Tyr Trp 385 390 395 400
Arg Met Arg Pro Leu Phe Gin Glu Leu Leu Gly Asn His Ala Arg Cys 405 410 415
Pro Tyr Arg Ala Leu Leu Arg Thr His Cys Pro Leu Arg Ala Met Ala 420 425 430
Ala Lys Glu Gly Ser Gly Asn Gin Ala His Arg Gly Val Gly Tie Cys 435 440 445
Pro LeU GlU Arg Pro Val Ala Ala Pro Gin Glu Gin Thr Asp Ser Thr 450 455 460
Arg Leu Val Gin Leu Leu Arg Gin His Ser Ser Pro Trp Gin Val Tyr 465 470 475 480
Ala Phe Leu Arg Ala Cys Leu Cys Arg Leu Val Pro Ala Gly Leu Trp 485 490 495
Gly Ser Gly His Asn Arg Arg Arg Phe Leu Arg Asn Val Lys Lys Phe 500 505 510
Val Ser Leu Gly Lys His Ala Lys Leu Ser Leu Gin Glu Leu Thr Trp 515 520 525
Lys Met Arg Val Gin Asp Cys Ala Trp Leu Arg Gly Ser Pro Gly Ala 530 535 540
Arg Cys Val Pro Ala Ala Glu His Arg Arg Arg Glu Glu Val Leu Ala 545 550 555 560
Lys Leu Leu Cys Trp Leu Met Gly Thr Tyr Val Val Glu Leu Leu Lys 565 570 575
Ser Phe Phe Tyr Val Thr Glu Thr Thr Phe Gin Lys Asn Arg Leu Phe 580 585 590
Phe Tyr Arg Lys Arg Ile Trp Ser Gin Leu Gin Ser Ile Gly Ile Arg 595 600 605
Gin His Phe Asn Ser Val His Leu Arg Glu Leu Ser Glu Ala Glu Val 610 615 620
Arg Arg His Gin Glu Ala Arg Pro Thr Leu Leu Thr Ser Lys Leu Arg 625 630 635 640
Phe Leu Pro Lys Pro Ser Gly Leu Arg Pro Ile Val Asn Met Asp Tyr 645 650 655
Val Val Gly Ala Arg Thr Phe Arg Arg Asp Lys Lys Val Arg His Leu 660 665 670
Thr Ser Gin Val Lys Asn Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala 675 680 685
Arg Arg Pro Ser Leu Leu Gly Ala Ser Val Leu Gly Met Asp Asp Ile 690 695 700
His Arg Val Trp Arg Ser Phe Val Leu Arg Val Arg Ala Gin Asp Pro 705 710 715 720
Ala Pro Gin Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp 725 730 735
Ala Leu Pro Gin Asp Lys Leu Val Glu Val Ile Ala Asn Val Ile Arg 740 745 750
Pro Gin Glu Asn Thr Tyr Cys Val Arg His Tyr Ala Val Val Gin Arg 755 760 765
Thr Ala Gin Gly His Val Arg Lys Ser Phe Lys Arg His Val Ser Thr 770 775 780
Phe Val Asp Leu Gin Pro Tyr Met Arg Gin Phe Val Glu His Leu Gin 785 790 795 800
Glu Thr Ser Ser Leu Arg Asp Ala Val Val Ile Glu Gin Ser Ser Ser 805 810 815
Leu Asn Glu Thr Gly His Ser Leu Phe His Leu Phe Leu Arg Leu Val 820 825 830
His Asn His Val Ile Arg Ile Gly Gly lys Ser Tyr Val Gin Cys Gin 835 840 845
Gly Ile Pro Gin Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys 850 855 860
Tyr Gly Asp Met Glu Ser Arg Leu Phe Ser Gly Ile Gin Gin Asp Gly 865 870 875 880
Val Leu Leu Arg Leu Phé Léu Leu Val Thr Pro His Leu Ala Gin Ala 885 890 895
Gin Ala Phe Leu Arg Thr Leu Val Ser Gly Val Pro Glu Tyr Gly Cys 900 905 910
Thr Ala Asn Leu Gin Lys Thr Ala Val Asn Phe Pro Val Asp Thr Gly 915 920 925
Ala Pro Gly Ser Ala Ala Pro Leu Gin Leu Pro Ala His Cys Leu Phe 930 935 940
Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Phe Cys 945 950 955 960
Asp Tyr Ser Ser Tyr Ala Gin Thr Ser Ile Arg Ser Ser Leu Thr Phe 965 970 975
Ser Gin Gly Thr Arg Pro Gly Arg Asn Met Arg Arg Lys Leu Leu Ala 980 985 990
Val Met Arg Leu Lys Cys Cys Ala Val Phe Leu Asp Leu Gin Val Asn 995 1000 1005
Ser Ile His Thr Val Tyr Thr Asn Ile Tyr Lys Ile Phe Leu Leu 1010 1015 1020
Gin Ala Tyr Arg Phe His Ala Cys Val Leu Gin Phe Pro Phé Asn 1025 1030 1035
Gin Pro Val Arg Lys Asn Pro Ser Phe Phe Leu Arg Val Ile Ala 1040 1045 1050
Asp Thr Ala Ser Arg Cys Tyr Ser Leu Leu Lys Ala Lys Asn Thr 1055 1060 1065
Gly Leu Ser Leu Gly Ala Lys Gly Ala Ser Gly Pro Phe Pro Ser 1070 1075 1080
Glu Ala Ala Arg Trp Leu Cys Leu His Ala Phe Leu Leu Lys Leu 1085 1090 1095
Ala Arg His Ser Ser Thr Tyr Arg Cys Leu Leu Gly Pro Leu Arg 1100 1105 1110
Ala Ala Lys Ala His Leu Ser Arg Gin Leu Pro Arg Gly Thr Leu 1115 1120 1125
Ala Ala Leu Glu Ala Ala Ala Asp Pre Ser Leu Thr Ala Asp Phe 1130 1135 1140
Lys Thr lie Leu Asp Thr Gia Leu Lys Leu Ser Asp Tyr Glu Gly 1145 1150 1155
Arg Leu lie Gin Asn Ser Leu Thr Gly Lys Pro He Pro Asn Pro 1160 1165 1170
Leu Leu Gly Leu Asp Ser Thr 1175 1180 <210> 5 <211 > 1123
<212> PRT <213> Canis familiaris <400>5
Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ala Leu Leu Arg Gly 15 10 15
Arg Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Leu Arg Arg Leu Gly 20 25 30
Pro Pro Gly Arg Leu Leu Val Arg Arg Gly Asp Pro Ala Ala Phe Arg 35 40 45
Ala Leu Val Ala Gin Cys Leu Val Cys Val Pro Trp Gly Ala Arg Pro 50 55 60
Pro Pro Ala Ala Pro Cys Phe Arg Gin Val Ser Cys Leu Lys Glu Leu 65 70 75 80
Val Ala Arg Val Val Gin Arg Leu Cys Glu Arg Gly Ala Arg Asn Val 85 90 95
Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro 100 105 110
Val Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr 115 120 125
Glu Thr Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val 130 135 140
Gly Asp Asp Val Leu Thr His Leu Leu Ala Arg Cys Ala Leu Tyr Leu 145 150 155 160
Leu Val Ala Pro Ser Cys Ala Tyr Gin Val Cys Gly Pro Pro Leu Tyr 165 170 175
Asp Leu Cys Ala Pro Ala Ser Leu Pro Leu Pro Ala Pro Gly Leu Pro 180 185 190
Gly Leu Pro Gly Leu Pro Gly Leu Gly Ala Gly Ala Gly Ala Ser Ala 195 200 205
Asp Leu Arg Pro Thr Arg Gin Ala Gin Asn Ser Gly Ala Arg Arg Arg 210 215 220
Arg Gly Set Pro Gly Ser Gly Val Pro Leu Ala Lys Arg Pro Arg Arg 225 230 235 240
Ser Val Ala Ser Glu Pro Glu Arg Gly Ala His Arg Ser Phe Pro Arg 245 250 255
Ala Gin Gin Pro Pro Val Ser Glu Ala Pro Ala Val Thr Pro Ala Val 260 265 270
Ala Ala Ser Pro Ala Ala Ser Trp Glu Gly Gly Pro Pro Gly Thr Arg 275 280 285
Pro Thr Thr Pro Ala Trp His Pro Tyr Pro Gly Pro Gin Gly Val Pro 290 295 300
His Asp Pro Ala His Pro Glu Thr Lys Arg Phe Leu Tyr Cys Ser Gly 305 310 315 320
Gly Arg Glu Arg Leu Arg Pro Ser Phe Leu Leu Ser Ala Leu Pro Pro 325 330 335
Thr Leu Ser Gly Ala Arg Lys Leu val Glu Thr lie Phe Leu Gly Ser 340 345 350
Ala Pro Gin Lys Pro Gly Ala Ala Arg Arg Met Arg Arg Leu Pro Ala 355 360 365
Arg Tyr Trp Arg Met Arg Pro Leu Phe Gin Glu Leu Leu Gly Asn His 370 375 380
Ala Arg Cys Pro Tyr Arg Ala Leu Leu Arg Thr His Cys Pro Leu Arg 385 390 395 400
Ala Met Ala Ala Lys Glu Gly Ser Gly Asn Gin Ala His Arg Gly Val 405 410 415
Gly He Cys Pro Leu Glu Arg Pro Val Ala Ala Pro Gin Glu Gin Thr 420 425 430
Asp Ser Thr Arg Leu Val Gin Leu Leu Arg Gin His Ser Ser Pro Trp 435 440 445
Gin Val Tyr Ala Phe Leu Arg Ala Cys Leu Cys Trp Leu Val Pro Thr 450 455 460
Gly Leu Trp Gly Ser Arg His Asn Gin Arg Arg Phe Leu Arg Asn Val 465 470 475 480
Lys Lys Phe He Ser Leu Gly Lys His Ala Lys Leu Ser Leu Gin Glu 485 490 495
Leu Thr Trp Lys Met Lys Val Arg Asp Cys Thr Trp Leu His Gly Asn 500 505 510
Pro Gly Ala Cys Cys Val Pro Ala Ala Glu His Arg Arg Arg Glu Glu 515 520 525 lie Leu Ala Arg Phe Leu Val Leu Val Asp Gly His lie Tyr Val Val 530 535 540
Lys Leu Leu Arg Ser Phe Phe Tyr Val Thr Glu Thr Thr Phe Gin Lys 545 550 555 560
Asn Arg Léu Phe Phe Tyr Arg Lys Ser Val Trp Ser Gin Leu Gin Ser 565 570 575
He Gly He Arg Gin Leu Phe Asn Ser Val His Leu Arg Glu Léu Ser 580 585 590
Glu Ala Glu val Arg Arg His Arg Glu Ala Arg Pro Ala Leu Leu Thr 595 600 605
Ser Arg LeU Arg Phe Leu Pro Lys Pro Ser Gly Leu Arg Pro He Val 610 615 620
Asn Met Asp Tyr He Met Gly Ala Arg Thr Phe His Arg Asp Lys Lys 625 630 635 640
Val Gin His Leu Thr Ser Gin Leu Lys Thr Leu Phe Ser Val Leu Asn 645 650 655
Tyr Glu Arg Ala Arg Arg Pro Ser Leu Leu Gly Ala Ser Met Leu Gly 660 665 670
Met Asp Asp He His Arg Ala Trp Arg Thr Phe Val Leu Arg lie Arg 675 680 685
Ala Gin Asn Pro Ala Pro Gin Leu Tyr Phe Val Lys Val Asp Val Thr 690 695 700
Gly Ala Tyr Asp Ala Leu Pro Gin Asp Arg Leu Val Glu Val He Ala 705 710 715 720
Asn Val He Arg Pro Gin Glu Ser Thr Tyr Cys Val Arg His Tyr Ala 725 730 735
Val Val Gin Arg Thr Ala Arg Gly His Val Arg Lys Ala Phe Lys Arg 740 745 750
His Val Ser· Thr Phe Ala Asp Leu Gin Pro Tyr Met Arg Gin Phe Val 755 760 765
Glu Arg Leu Gin Glu Thr Ser Leu Leu Arg Asp Ala Val Val lie Glu 770 775 780
Gin Ser Ser Ser Leu Asn Glu Ala Gly Ser Ser Leu Phe His Leu Phe 785 790 795 800
Leu Arg Leu Val His Asn His Val Val Arg Ile Gly Gly Lys Ser Tyr 805 810 815
Ile Gin Cys Gin Gly Val Pro Gin Gly Ser Ile Leu Ser Thr Leu Leu 820 825 830
Cys Ser Leu Cys Tyr Gly Asp Met Glu Arg Arg Leu Phe Pro Gly Ile 835 840 845
Glu Gin Asp Gly Val Leu Leu Arg Leu Val Asp Asp Phe Leu Leu Val 850 855 860
Thr Pro His Leu Thr Gin Ala Gin Ala Phe Leu Arg Thr Leu Val Lys 865 870 875 880
Gly Val Pro Glu Tyr Gly Cys Arg Ala Asn Leu Gin Lys Thr Ala Val 835 890 895
Asn Phe Pro Val Glu Asp Gly Ala Leu Gly Ser Ala Ala Pro Leu Gin 900 905 910
Leu Pro Ala His Cys Leu Phe Pro Trp Cys Gly Leu Leu Leu Asp Thr 915 920 925
Arg Thr Leu Glu Val Ser Cys Asp Tyr Ser Ser Tyr Ala His Thr Ser 930 935 940
Ile Arg Ala Ser Leu Thr Phe Ser Gin Gly Ala Lys Pro Gly Arg Asn 945 950 955 960
Met Arg Arg Lys Leu Leu Ala val Leu Arg Leu Lys Cys Cys Ala Leu 965 970 975
Phe Leu Asp Leu Gin Val Asn Gly Ile His Thr Val Tyr Met Asn Val 980 985 990
Tyr Lys Ile Phe Leu Leu Gin Ala Tyr Arg Phe His Ala Cys Val Leu 995 1000 1005
Gin Leu Pro Phe Asn Gin Pro Val Arg Lys Asn Pro Ser Phe Phe 1010 1015 1020
Leu Arg Val Ile Ala Asp Thr Ala Ser Cys Cys Tyr Ser Leu Leu 1025 1030 1035
Lys Ala Arg Asn Ala Gly Leu Ser Leu Gly Ala Lys Gly Ala Ser 1040 1045 1050
Gly Leu Phe Pro Ser Glu Ala Ala Arg Trp Leu Cys Leu His Ala 1055 1060 1065
Phe Leu Leu Lys Leu Ala His His Ser Gly Thr Tyr Arg Cys Leu 1070 1075 1080
Leu Giv Ala Leu Gin Ala Ala Lvs Ala His Leu Ser Ara Gin Leu 1085 1090 “ 1095
Pro Arg Gly Thr Leu Ala Ala Leu Glu Ala Ala Ala Asp Pro Ser 1100 1105 1110
Leu Thr Ala Asp Phe Lys Thr Ile Leu Asp 1115 1120 <210> 6 <211>1073
<212> PRT <213> Canis familiaris <400> 6
Arg Ala Leu Val Ala Gin Cys Leu Val Cys Val Pro Trp Gly Ala Arg 1 5 10 15
Pro Pro Pro Ala Ala Pro Cys Phe Arg Gin Val Ser Cys Leu Lys Glu 20 25 30
Leu Val Alå Arg Val Val Gin Arg Leu Cys Glu Arg Gly Ala Arg Asn 35 40 45
Val Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro 50 55 60
Pro Val Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro ASn Thr Val 65 70 75 80
Thr Glu Thr Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg 85 90 95
Val Gly Asp Asp Val Leu Thr His Leu Leu Ala Arg Cys Ala Leu Tyr 100 105 110
Leu Leu Val Ala Pro Ser Cys Ala Tyr Gin Val Cys Gly Pro Pro Leu 115 120 125
Tyr Asp Leu Cys Ala Pro Ala Ser Leu Pro Leu Pro Ala Pro Gly Leu 130 135 140
Pro Gly Leu Pro Gly Leu Pro Gly Leu Gly Ala Gly Ala Gly Ala Ser 145 150 155 160
Ala Asp Leu Arg Pro Thr Arg Gin Ala Gin Asn Ser Gly Ala Arg Arg 165 170 175
Arg Arg Gly Ser Pro Gly Ser Gly Val Pro Leu Ala Lys Arg Pro Arg 180 185 190
Arg Ser Val Ala Ser Glu Pro Glu Arg Gly Ala His Arg Ser Phe Pro 195 200 205
Arg Ala Gin Gin Pro Pro Val Ser Glu Ala Pro Ala Val Thr Pro Ala 210 215 220
Val Ala Ala Ser Pro Ala Ala Ser Trp Glu Gly Gly Pro Pro Gly Thr 225 230 235 240
Arg Pro Thr Thr Pro Ala Trp His Pro Tyr Pro Gly Pro Gin Gly Val 245 250 255
Pro His Asp Pro Ala His Pro Glu Thr Lys Arg Phe Leu Tyr Cys Ser 260 265 270
Gly Gly Arg Glu Arg Leu Arg Pro Ser Phe Leu Leu Ser Ala Leu Pro 275 280 285
Pro Thr Leu Ser Gly Ala Arg Lys Leu Val Glu Thr lie Phe Leu Gly 290 295 300
Ser Ala Pro Gin Lys Pro Gly Ala Ala Arg Arg Met Arg Arg Leu Pro 305 310 315 320
Ala Arg Tyr Trp Arg Met Arg Pro Leu Phe Gin Glu Leu Leu Gly Asn 325 330 335
His Ala Arg Cys Pro Tyr Arg Ala Leu Leu Arg Thr His Cys Pro Leu 340 345 350
Arg Ala Met Ala Ala Lys Glu Gly Ser Gly Asn Gin Ala His Arg Gly 355 360 365
Val Gly He Cys Pro Leu Glu Arg Pro Val Ala Ala Pro Gin Glu Gin 370 375 380
Thr Asp Ser Thr Arg Leu Val Gin Leu Leu Arg Gin His Ser Ser Pro 385 390 395 400
Trp Gin Val Tyr Ala Phe Leu Arg Ala Cys Leu Cys Trp Leu Val Pro 405 410 415
Thr Gly Leu Trp Gly Ser Arg His Asn Gin Arg Arg Phe Leu Arg Asn 420 425 430
Val Lys Lys Phe He Ser Leu Gly Lys His Ala Lys Leu Ser Leu Gin 435 440 445
Glu Leu Thr Trp Lys Met Lys Val Arg Asp Cys Thr Trp Leu His Gly 450 455 460
Asn Pro Gly Ala Cys Cys Val Pro Ala Ala Glu His Arg Arg Arg Glu 465 470 475 480
Glu He Leu Ala Arg Phe Leu Val Leu Val Asp Gly His He Tyr Val 485 490 495
Val Lys Leu Leu Arg Ser Phe Phe Tyr Val Thr Glu Thr Thr Phe Gin 500 505 510
Lys Asn Arg Leu Phe Phe Tyr Arg Lys Ser Val Trp Ser Gin Leu Gin 515 520 525
Ser Ile Gly Ile Arg Gin Leu Phe Asn Ser Val His Leu Arg Glu Leu 530 535 540
Ser Glu Ala Glu Val Arg Arg His Arg Glu Ala Arg Pro Ala Leu Leu 545 550 555 560
Thr Ser Arg Leu Arg Phe Leu Pro Lys Pro Ser Gly Leu Arg Pro Ile 565 570 575
Val Asn Met Asp Tyr Ile Met Gly Ala Arg Thr Phe His Arg Asp Lys 580 585 590
Lys Val Gin His Leu Thr Ser Gin Leu Lys Thr Leu Phe Ser Val Leu 595 600 605
Asn Tyr Glu Arg Ala Arg Arg Pro Ser Leu Leu Gly Ala Ser Met Leu 610 615 620
Gly Met Asp Asp Ile His Arg Ala Trp Arg Thr Phe Val Leu Arg Ile 625 630 635 640
Arg Ala Gin Asn Pro Ala Pro Gin Leu Tyr Phe Val Lys Val Asp Val 645 650 655
Thr Gly Ala Tyr Asp Ala Leu Pro Gin Asp Arg Leu Val Glu Val Ile 660 665 670
Ala Asn Val Ile Arg Pro Gin Glu Ser Thr Tyr Cys Val Arg His Tyr 675 680 685
Ala val val Gin Arg Thr Ala Arg Gly His val Arg Lys Ala Phe Lys 690 695 700
Arg HiS Val Ser Thr Phe Ala Asp Leu Gin Pro Tyr Met Arg Gin Phe 705 710 715 720
Val Glu Arg Leu Gin Glu Thr Ser Leu Leu Arg Asp Ala Val Val Ile 725 730 735
Glu Gin Ser Ser Ser Leu Asn Glu Ala Gly Ser Ser Leu Phe His Leu 740 745 750
Phe Leu Arg Leu Val His Asn His Val Val Arg Ile Gly Gly Lys Ser 755 760 765
Tyr Ile Gin Cys Gin Gly Val Pro Gin Gly Ser Ile Leu Ser Thr Leu 770 775 780
Leu CyS Ser Leu Cys Tyr Gly Asp Met Glu Arg Arg Leu Phe Pro Gly 785 790 795 800
Ile Glu Gin Asp Gly Val Leu Leu Arg Leu Phe Leu Leu Val Thr Pro 805 810 815
His Leu Thr Gin Ala Gin Ala Phe Leu Arg Thr Leu Val Lys Gly Val 820 825 830
Pro Glu Tyr Gly Cys Arg Ala Asn Leu Gin Lys Thr Ala Val Asn Phe 835 840 845
Pro Val Glu Asp Gly Ala Leu Gly Ser Ala Ala Pro Leu Gin Leu Pro 850 855 860
Ala His Cys Leu Phe Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr 865 870 875 880
Leu Glu Val Ser Cys Asp Tyr Ser Ser Tyr Ala His Thr Ser lie Arg 885 890 895
Ala Ser Leu Thr Phe Ser Gin Gly Ala Lys Pro Gly Arg Asn Met Arg 900 905 910
Arg Lys Leu Leu Ala Val Leu Arg Leu Lys Cys Cys Ala Leu Phe Leu 915 920 925
Asp Leu Gin Val Asn Gly lie His Thr Val Tyr Met Asn Val Tyr Lys 930 935 940
He Phe Leu Leu Gin Ala Tyr Arg Phe His Ala Cys Val Leu Gin Leu 945 950 955 960
Pro Phe Asn Gin Pro Val Arg Lys Asn Pro Ser Phe Phe Leu Arg Val 965 970 975
He Ala Asp Thr Ala Ser Cys Cys Tyr Ser Leu Leu Lys Ala Arg Asn 980 985 990
Ala Gly Leu Ser Leu Gly Ala Lys Gly Ala Ser Gly Leu Phe Pro Ser 995 1000 1005
Glu Ala Ala Arg Trp Leu Cys Leii His Ala Phe Leu Leu Lys Léu 1010 1015 1020
Ala His His Ser Gly Thr Tyr Arg Cys Leu Leu Gly Ala Leu Gin 1025 1030 1035
Ala Ala Lys Ala His Leu Ser Arg Gin Leu Pro Arg Gly Thr Leu 1040 1045 1050
Ala Ala Leu Glu Ala Ala Ala Asp Pro Ser Leu Thr Ala Asp Phe 1055 1060 1065
Lys Thr lie Leu Asp 1070 <210> 7 <211>1024
<212> PRT <213> Felis catus <400> 7
Asn vsi hen 4ia Fne u±y vne A±a i«u neu asp u±y &amp;ia arg uxy isxy 15 10 15
Pro Pro Val Val Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr 20 25 30
Val Thr Glu Thr Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg 35 40 45
Arg Val Gly Asp Asp Val Leu Ala His Leu Leu Thr Arg Cys Ala Leu 50 55 60
Tyr Val Leu Val Ala Pro Ser Cys Ala Tyr Gin Val Cys Gly Pro Pro 65 70 75 80
Leu Tyr Asp Leu Cys Ala Pro Ala Ala Thr Arg Pro Leu Ala Thr Ser 85 90 95
Gly His Arg Pro Gly Thr Arg Met Asp Leu Arg Pro Thr Arg Gin Ala 100 105 110
Arg Asn Ala Gly Ala Arg Arg Arg Arg Gly Ala Gly Gly Ser Ser Pro 115 120 125
Pro Leu Ala Lys Arg Pro Arg His Asp Val Lys Thr Pro Glu Pro Glu 130 135 140
Arg Gly Pro Ala Ser Pro Ser Ser Arg His Pro Pro Gly Arg Ala His 145 150 155 160
Gly Leu Ser Gly Gly Glu Pro Gly Ala Val Thr Ser Ala Arg Ala Ala 165 170 175
Ala Glu Ala Asn Ser Gly Glu Gly Gly Pro Pro Gly Thr Arg Leu Thr 180 185 190
Ser Ala Gly Ala Gin Leu Ser Arg Pro Gin Gly Val Pro Leu Ser His 195 200 205
Leu Ser His Pro Glu Thr Lys His Phe Leu Tyr Cys Pro Gly Gly Lys 210 215 220
Glu Arg Leu Arg Pro Ser Phe Leu Leu Ser Ala Leu Arg Pro Ser Leu 225 230 235 240
Thr Gly Ala Arg Thr Leu Leu Glu Ala Ile Phe Leu Gly Ser Lys Ser 245 250 255
Pro Arg Pro Gly Ala Ala Arg Arg Thr Arg Arg Leu Pro Ala Arg Tyr 260 265 270
Trp Arg Met Arg Pro Leu Phe Arg Glu Leu Leu Ala Asn His Ala Arg 275 280 285
Cys Pro Tyr Asp Ala Leu Leu Arg Thr His Cys Pro Leu Arg Ala Pro 290 295 300
Ala Pro Ala Glu Gly Ser Ser Arg Gly Val Gly Gly Gly Ala Gly Gly 305 310 315 320
Cys Ala Leu Gly Arg Pro Pro Gly Ala Pro Gin Glu Gin Thr Asp Ser 325 330 335
Thr Arg Leu Val Gin Leu Leu Arg Gin His Ser Ser Pro Trp Gin Val 340 345 350
Tyr Ala Phe Leu Arg Ala Cys Leu Cys Arg Leu Val Pro Ala Gly Leu 355 360 365
Trp Gly Ser Gly His Asn Arg Arg Arg Phe Leu Arg Asn Val Lys Lys 370 375 380
Phe Val Ser Leu Gly Lys His Ala Lys Leu Ser Leu Gin Glu Leu Thr 385 390 395 400
Trp Lys Met Arg Val Gin Asp Cys Ala Trp Leu Arg Gly Ser Pro Gly 405 410 415
Ala Arg Cys Val Pro Ala Ala Glu His Arg Arg Arg Glu Glu Val Leu 420 425 430
Ala Lys Leu Leu Cys Trp Leu Met Gly Thr Tyr Val Val Glu Leu Leu 435 440 445
Lys Ser Phe Phe Tyr Val Thr Glu Thr Thr Phe Gin Lys Asn Arg Leu 450 455 460
Phe Phe Tyr Arg Lys Arg lie Trp Ser Gin Leu Gin Ser He Gly He 465 470 475 480
Arg Gin His Phe Asn Ser Val His Leu Arg Glu Leu Ser Glu Ala Glu 485 490 495
Val Arg Arg His Gin Glu Ala Arg Pro Thr Leu Leu Thr Ser Lys Leu 500 505 510
Arg Phe Leu Pro Lys Pro Ser Gly Leu Arg Pro He Val Asn Met Asp 515 520 525
Tyr Val Val Gly Ala Arg Thr Phe Arg Arg Asp Lys Lys Val Arg His 530 535 540
Leu Thr Ser Gin Val Lys Asn Leu Phe Ser Val Leu Asn Tyr Glu Arg 545 550 555 560
Ala Arg Arg Pro Ser Leu Leu Gly Ala Ser Val Leu Gly Met Asp Asp 565 570 575 lie His Arg Val Trp Arg Ser Phe Val Leu Arg Val Arg Ala Gin Asp 580 585 590
Pro Ala Pro Gin Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr 595 600 605
Asp Ala Leu Pro Gin Asp Lys Leu Val Glu Val lie Ala Asn Val He 610 615 620
Arg Pro Gin Glu Asn Thr Tyr Cys Val Arg His Tyr Ala Val Val Gin 625 630 635 640
Arg Thr Ala Gin Gly His Val Arg Lys Ser Phe Lys Arg His Val Ser 645 650 655
Thr Phe Val Asp Leu Gin Pro Tyr Met Arg Gin Phe Val Glu His Leu 660 665 670
Gin Glu Thr Ser Ser Leu Arg Asp Ala Val Val He Glu Gin Ser Ser 675 680 685
Ser Leu Asn Glu Thr Gly His Ser Leu Phe His Leu Phe Leu Arg Leu 690 695 700
Val His Asn His Val He Arg He Gly Gly Lys Ser Tyr Val Gin Cys 705 710 715 720
Gin Gly lie Pro Gin Gly Ser lie Leu Ser Thr Leu Leu Cys Ser Leu 725 730 735
Cys Tyr Gly Asp Met Glu Ser Arg Leu Phe Ser Gly He Gin Gin Asp 740 745 750
Gly Val Leu Leu Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His 755 760 765
Leu Ala Gin Ala Gin Ala Phe Leu Arg Thr Leu Val Ser Gly Val Pro 770 775 780
Glu Tyr Gly Cys Thr Ala Asn Leu Gin Lys Thr Ala Val Asn Phe Pro 785 790 795 800
Val Asp Thr Gly Ala Pro Gly Ser Ala Ala Pro Leu Gin Leu Pro Ala 805 810 815
His Cys Leu Phe Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu 820 825 830
Glu Val Phe Cys Asp Tyr Ser Ser Tyr Ala Gin Thr Ser He Arg Ser 835 840 845
Ser Leu Thr Phe Ser Gin Gly Thr Arg Pro Gly Arg Asn Met Arg Arg 850 855 860
Lys Leu Leu Ala Val Met Arg Leu Lys Cys Cys Ala Val Phe Leu Asp 865 870 875 880
Leu Gin Val Asn Ser He His Thr Val Tyr Thr Asn He Tyr Lys He 885 890 895
Phe Leu Leu Gin Ala Tyr Arg Phe His Ala Cys Val Leu Gin Phe Pro 900 905 910
Phe Asn Gin Pro Val Arg Lys Asn Pro Ser Phe Phe Leu Arg Val lie 915 920 925
Ala Asp Thr Ala Ser Arg Cys Tyr Ser Leu Leu Lys Ala Lys Asn Thr 930 935 940
Gly Leu Ser Leu Gly Ala Lys Gly Ala Ser Gly Pro Phe Pro Ser Glu 945 950 955 960
Ala Ala Arg Trp Leu Cys Leu His Ala Phe Leu Leu Lys Leu Ala Arg 965 970 975
His Ser Ser Thr Tyr Arg Cys Leu Leu Gly Pro Leu Arg Ala Ala Lys 980 985 990
Ala Gin Leu Arg Arg Gin Leu Pro Arg Ala Thr Leu Asp Ala Leu Glu 995 1000 1005
Ala Ala Ala Ser Pro Gly Léu Pro Ala Asp Phe Arg Thr He Leu 1010 1015 1020
Asp <210> 8 <211>9
<212> PRT <213> Artificial <220> <223> H2-restricted peptide <400>8
Arg Pro He Val Asn Met Asp Tyr lie 1 5 <210> 9 <211>9
<212> PRT <213> Artificial <220> <223> H2-restricted peptide <400>9
Arg Gin Leu Phe Asn Ser Val His Leu 1 5 <210> 10 <211>9
<212> PRT <213> Artificial <220> <223> H2-restricted peptide <400> 10
Thr Val Tyr Met Asn Val Tyr Lys Ile 1 5 <210> 11 <211> 15
<212> PRT <213> Artificial <220> <223> H2-restricted peptide <400> 11
Cys Leu Leu Gly Pro Leu Arg Ala Ala Lys Ala His Leu Ser Arg 1 5 10 15 <210> 12 <211> 15
<212> PRT <213> Artificial <220> <223> H2-restricted peptide <400> 12
Arg Cys Léu Leu Gly Pro Leu Arg Ala Ala Lys Ala His Leu Ser 15 10 15 <210> 13 <211> 15
<212> PRT <213> Artificial <220> <223> H2-restricted peptide <400> 13
Tyr Ser Ser Tyr Ala Gin Thr Ser Ile Arg Ser Ser Leu Thr Phe 15 10 15 <210> 14 <211> 15
<212> PRT <213> Artificial <220> <223> H2-restricted peptide <400> 14
Gly Pro Leu Arg Ala Ala Lys Ala His Leu Ser Arg Gin Leu Pro 15 10 15
REFERENCES CITED IN THE DESCRIPTION
This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.
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Claims (14)

1. Immunogen sammensætning omfattende en nukleinsyre, der omfatter en sekvens, der koder for en hunde-telomerase-revers-transkriptase (TERT), hvor aminosyrerne VDD i hunde-TERT'en er slettet, hvilket tilvejebringer inaktivering af telomerase katalytisk aktivititet, og yderligere hvor de N-terminale 47-aminosyrer i forhold til fuldlængde hunde-TERT-sekvens er slettet.An immunogenic composition comprising a nucleic acid comprising a sequence encoding a canine telomerase reverse transcriptase (TERT) wherein the amino acids VDD in the canine TERT are deleted, providing inactivation of telomerase catalytic activity, and further wherein the N-terminal 47-amino acids relative to the full-length canine TERT sequence is deleted. 2. Sammensætning ifølge krav 1, hvor nævnte nukleinsyre yderligere koder for ubiquitin.The composition of claim 1, wherein said nucleic acid further encodes ubiquitin. 3. Sammensætning ifølge krav 1 eller 2, hvor nukleinsyren er et DNA-plasmid.The composition of claim 1 or 2, wherein the nucleic acid is a DNA plasmid. 4. Sammensætning ifølge krav 3, hvor nukleinsyren koder for et protein omfattende eller bestående af SEQ ID NO: 6.The composition of claim 3, wherein the nucleic acid encodes a protein comprising or consisting of SEQ ID NO: 6. 5. Nukleinsyre omfattende en sekvens, der koder for en hunde-telomerase-revers-transkriptase (TERT), hvor aminosyrerne VVD i hunde-TERT'en er slettet, hvilket tilvejebringer inaktivering af telomerase katalytisk aktivitet og yderligere hvor de N-terminale 47-aminosyrer i forhold til fuldlængde hunde-TERT sekvens er slettet.A nucleic acid comprising a sequence encoding a canine telomerase reverse transcriptase (TERT), wherein the amino acids VVD of the canine TERT are deleted, providing inactivation of telomerase catalytic activity and further wherein the N-terminal 47 amino acids relative to full-length canine TERT sequence are deleted. 6. Nukleinsyre ifølge krav 5, hvor nævnte nukleinsyre yderligere koder for ubiquitin.The nucleic acid of claim 5, wherein said nucleic acid further encodes ubiquitin. 7. Nukleinsyre ifølge krav 5 eller 6, som koder for en proteinsekvens bestående af SEQ ID NO: 2.The nucleic acid of claim 5 or 6, which encodes a protein sequence consisting of SEQ ID NO: 2. 8. Nukleinsyre ifølge krav 5 omfattende en sekvens valgt fra gruppen bestående af SEQ ID NO: 1 eller nukleotider 241-3459 af SEQ ID NO: 1.The nucleic acid of claim 5 comprising a sequence selected from the group consisting of SEQ ID NO: 1 or nucleotides 241-3459 of SEQ ID NO: 1. 9. Immunogen sammensætning ifølge et hvilket som helst af kravene 1 til 4, eller nukleinsyre ifølge et hvilket som helst af kravene 5 til 8, til anvendelse i forebyggelse eller behandling af en tumor i en hund.An immunogenic composition according to any one of claims 1 to 4, or nucleic acid according to any of claims 5 to 8, for use in the prevention or treatment of a tumor in a dog. 10. Immunogen sammensætning eller nukleinsyre til anvendelse i forebyggelse eller behandling af en tumor i en hund ifølge krav 9, hvor tumoren er en ondartet tumor.An immunogenic composition or nucleic acid for use in the prevention or treatment of a tumor in a dog according to claim 9, wherein the tumor is a malignant tumor. 11. Immunogen sammensætning eller nukleinsyre, til anvendelse i forebyggelse eller behandling af en tumor i en hund ifølge krav 9, hvor tumoren er valgt fra gruppen bestående af blærekræft, hjernetumor, mammatumorer og karcinom, mastcelletumorer, ondartet histiocytose og histiocytiske sarkomer, pladeepithelcarcinomer, hæmangiosarkom, lymfom, særligt B-celle lymfom, melanom, osteosarkom, testikeltumorer.An immunogenic composition or nucleic acid, for use in the prevention or treatment of a tumor in a dog according to claim 9, wherein the tumor is selected from the group consisting of bladder cancer, brain tumor, mammary tumors and carcinoma, mast cell tumors, malignant histiocytosis and histiocytic sarcomas, squamous cell carcinomas, hematoma carcinomas, , lymphoma, especially B-cell lymphoma, melanoma, osteosarcoma, testicular tumors. 12. Immunogen sammensætning eller nukleinsyre til anvendelse ifølge et hvilket som helst af kravene 9 til 11, hvor sammensætningen eller nukleinsyren skal administreres af intradermal eller intramuskulær vej.An immunogenic composition or nucleic acid for use according to any one of claims 9 to 11, wherein the composition or nucleic acid is to be administered by intradermal or intramuscular route. 13. Immunogen sammensætning eller nukleinsyre til anvendelse ifølge et hvilket som helst af kravene 9 til 12, hvor hunden er i risiko for at udvikle en tumor, eller hvor hunden er rask, men gammel.An immunogenic composition or nucleic acid for use according to any one of claims 9 to 12, wherein the dog is at risk of developing a tumor or wherein the dog is healthy but old. 14. Immunogen sammensætning eller nukleinsyre til anvendelse ifølge et hvilket som helst af kravene 9 til 13, hvor sammensætningen eller nukleinsyren inducerer en langtidshukommelses immunrespons.An immunogenic composition or nucleic acid for use according to any one of claims 9 to 13, wherein the composition or nucleic acid induces a long-term memory immune response.
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